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https://f1000research.com/articles/7-714/v1	08 Jun 18	{ "type": "Systematic Review", "title": "Therapeutic interventions for acute complete ruptures of the ulnar collateral ligament of the thumb: a systematic review", "authors": [ "Mark Mikhail", "Justin C. R. Wormald", "Neal Thurley", "Nicholas Riley", "Benjamin J. F. Dean", "Mark Mikhail", "Justin C. R. Wormald", "Neal Thurley", "Nicholas Riley" ], "abstract": "Background: The aim of this study was to evaluate the effectiveness of interventions for acute complete rupture of the ulnar collateral ligament (UCL) of the thumb in adults. Methods: The following databases were searched: MEDLINE and EMBASE via OVID, CINAHL and SPORTDiscus via EBSCO, from database inception to 31st January 2018. Inclusion criteria were: (i) randomised controlled clinical trials (RCTs) or study of intervention with a comparator; (ii) participants with diagnosis of acute complete rupture of the UCL of the thumb; (iii) participants aged 18 years of age or older at enrolment; and (iv) published in a peer-reviewed English-language journal. Results: In total, six studies were identified for inclusion after screening. All studies had a high risk of bias. Three studies were retrospective comparative case series which compared two different surgical techniques (bone anchor versus pull out suture, suture versus pull out suture, suture versus steel wire). Of these studies, three were RCTs, two of which compared different rehabilitation regimes in patients managed surgically (plaster versus early mobilization, new spica versus standard spica). The remaining RCT compared two different rehabilitation regimes in a mixed group of surgically/non-surgically treated patients. The RCT comparing a standard spica with a new spica demonstrated a statistically significant improvement in outcomes with the new spica at all time points (range of motion, Dreiser index and VAS); this was also the only study to provide sufficient outcome data for further analysis. Conclusion: There is no prospective evidence comparing surgery to non-operative treatment for acute complete ruptures of the ulnar collateral ligament of the thumb. There is weak evidence to suggest that early mobilisation may be beneficial following surgical repair. Further research is necessary to better define which patients benefit from which specific interventions.", "keywords": [ "ulnar collateral ligament", "thumb", "rupture", "surgery" ], "content": "Introduction\n\nAcute complete ruptures of the ulnar collateral ligament (UCL) of the thumb are common injuries, accounting for around 50 in 100,000 presentations to Accident and Emergency departments. There is controversy as how to manage complete ruptures of the UCL best, although there is a degree of consensus regarding the broader treatment algorithm and general agreement that ‘true’ Stener lesions should be managed operatively1,2. The rate of the Stener lesion varies widely in the literature, perhaps reflecting the lack of reliability and accuracy of the various methods of diagnosis3,4.\n\nPatients should be assessed clinically to determine the degree of instability of the metacarpophalangeal joint (MCPJ) in both extension and 30° of flexion to test both proper and accessory collateral ligaments5. There is some evidence to suggest that the greater the instability the higher the chances are that a Stener lesion is present6,7. While there is evidence to support both the use of ultrasound and MRI, the latter appears slightly superior in terms of sensitivity and specificity8–10. A recent study by Stoop et al. investigated which factors predict the chances of surgery in UCL injuries11. It was found that not only did patient characteristics influence the chances of surgery, but that the individual surgeon’s preference was also predictive.\n\nOur aim was to perform a systematic review of the effectiveness of available interventions for acute complete rupture of the ulnar collateral ligament of the thumb in terms of patient-reported outcome measures and to assess the rates of adverse outcomes associated with these interventions.\n\n\nMethods\n\nThe systematic review was developed in accordance with the PRISMA statement (Supplementary File 1 contains a completed PRISMA checklist), using methodology decribed in the Cochrane Handbook for Systematic Reviews of Interventions. The protocol was developed prospectively and peer reviewed locally before registration on the PROSPERO database (CRD42018087656).\n\nA comprehensive search strategy was created in collaboration with a research librarian (N.T.) and was designed to capture all relevant articles pertaining to inventions for acute complete ruptures of the ulnar collateral ligament of the thumb (Supplementary File 2). The full search strategy is detailed on the PROSPERO website. The search strategy was applied to the following bibliographic databases from database inception until 31st January 2018: MEDLINE and EMBASE via OVID, CINAHL and SPORTDiscus via EBSCO from database inception until 31st January 2018.\n\nThe inclusion and exclusion criteria were defined prospectively during the protocol stage. Any study relating to acute complete ruptures of the ulnar collateral ligament of the thumb MCPJ in adults was included. Studies had to contain an intervention and a comparator (i.e. both non-randomised controlled trials, and randomised controlled trials, including semi/quasi randomised, cluster randomised trials and comparative case series). Any therapeutic intervention or control treatments were included.\n\nDuplicates were removed and relevant studies identified from the search were imported into Covidence for screening. Studies were independently screened by title and abstract by two authors (B.J.F.D. and M.M.). This was followed by a full-text evaluation of the selected studies from the first selection step these authors. Disagreement between the two reviewers was solved by consensus involving a third author (J.C.R.W.).\n\nTwo reviewers (M.M. and B.J.F.D) independently extracted data. Data was extracted using a custom data extraction sheet in Covidence. Any inconsistencies between the two reviewers’ forms were resolved by consensus discussion. A third review (J.C.R.W.) was available for any disagreement that could not be resolved by this initial discussion.\n\nIf data was not available from full-text articles or trial registrations, authors were contacted to provide this information. If authors were not contactable as regards additional data, then this aspect of the study was excluded from the data synthesis. If contactable authors did not respond to initial requests, they were sent two subsequent reminders over a minimum of 6 weeks. If there was still no response for the additional data, then this aspect of the study was excluded from the data synthesis.\n\nIncluded studies were assessed for risk of bias by two independent raters (B.J.F.D. and M.M.) using the Cochrane Collaboration’s tool for assessing risk of bias in randomised trials12. This followed the description in the Cochrane Handbook for Systematic Review of Interventions, version 5.1 (Part 2: 8.5.1)12. Any disagreements between ratings were resolved by discussion between the raters. A third party (J.C.R.W.) was available in any case where disagreements persisted after discussion.\n\nDescriptive analysis was performed for all demographic, intervention and outcome data to facilitate narrative interpretation and comparison across studies. It was decided that a direct-comparison meta-analysis would only be performed if data was available for similar time-points, outcomes and interventions across two or more studies. As this was not possible with the identified studies, we conducted a narrative synthesis of the results based on the domains of interest.\n\n\nResults\n\nA total of 158 studies were identified by the search, after duplicates were removed. After screening by full-text, six studies were identified as eligible for inclusion (Figure 1). Of these, three were randomized controlled trials (RCTs), and three were retrospective comparative case series. The number of studies identified and excluded at each stage is detailed in Figure 1.\n\nStudy characteristics of the included trials including the interventions and comparators are provided in Table 1. Of the three randomised controlled trials, two assessed the outcomes of different rehabilitation regimes in patients who had been exclusively treated with surgery13,14. The remaining RCT assessed the outcome in patients managed both surgically and non-surgically, who were randomised to treatment with either a plaster cast or a functional splint15. All three retrospective comparative case series compared different surgical techniques in patients exclusively managed surgically16–18. Table 2 details the basic demographics of the intervention and comparator groups, as well as the details about the outcome data provided. The full details of all included studies and the forest plots are included within the supplementary material (Supplementary File 3–Supplementary File 15).\n\nUCL, ulnar collateral ligament; MCPJ, metacarpophalangeal joint.\n\nUCL, ulnar collateral ligament; MCPJ, metacarpophalangeal joint.\n\nThe study by Sollerman et al.15 compared a functional splint with plaster cast treatment in patients with complete UCL ruptures; patients were managed both surgically and non surgically. The authors reported no difference in MCPJ range of movement (ROM), grip strength and sick leave taken; however, the data provided were insufficient for any further analysis, such as a forest plot.\n\nThe RCT by Rocchi et al. compared the outcomes of operated patients treated with either a traditional standard thumb spica which immobilized the MCPJ or a new modified thumb spica which allowed early MCP motion14. At 12 months the new spica group had increased MCPJ ROM (standardized mean difference (SMD), −3.69; 95% confidence interval (CI), −2.46–−4.92, P<0.0001), a better Dreiser index (SMD, 1.65; 95%CI, 0.81–2.50; P=0.0001) and reduced pain VAS (SMD, 1.53; 95% CI, 0.70–2.35; P=0.0003). There was no statistically significant difference between groups in tip pinch strength at any time point. The RCT by Crowley et al. compared outcomes between patients treated with early active mobilization or plaster immobilization after being treated surgically with Mitek anchor repair13. The outcome data was not provided, meaning that any further analysis was not possible.\n\nThe retrospective comparative case series by Saetta et al. demonstrated a higher chance of an excellent functional result with suture repair versus steel wire, but this was not statistically significant (risk ratio, 1.19; 95% CI, 0.82–1.71); the other outcome data was incomplete and thus precluded further analysis. The retrospective case series by Lane demonstrated no statistically significant difference in the chances of a full versus partial recovery in ROM of the MCPJ, of a full versus partial recovery in strength and of a full versus partial functional recovery17. The study by Katolik et al. did not provide adequate data with which to conduct any further analysis16.\n\nRocchi et al14. demonstrated no statistically significant difference in complication rate between treatment with the standard spica and the new spica (risk ratio, 1.5; 95% CI, 0.29–7.73); the complications consisted of three cases of temporary dysaesthesia and two cases of inflammatory scars. The complication rate was identical in both the early active mobilization and plaster cast groups in the study by Crowley et al13. (Risk ratio: 1.0, 95% CI: 0.32, 3.10); all six complications in this study were that of scar tethering, with all resolving with ultrasound therapy and massage. The studies by Saetta et al.17 and Sollerman et al.18 did not make any mention of specific complications. Lane17 demonstrated no statistically significant difference in the complication rate between the older method of pull out suture plus K-wire fixation and the new method of suture repair (risk ratio, 3.57; 95% CI, 0.25–50.15); there was one complication with the traditional method (broken pull-out suture at 2 weeks) and one with the new method (re-rupture at 9 months) The study by Katolik et al16. demonstrated a higher complication rate with pull-out suture versus bone anchor repair, but this was not statistically significant (risk ratio, 4.00; 95% CI, 0.92–17.30); all the ten complications were soft-tissue-related (five were persistent wound erythema consistent with wound infection and five were paraesthesiae, which resolved over time).\n\nAll criteria were judged as low, high or unclear risk of bias. Overall, all studies were deemed to be at a high risk of bias, particularly in terms of blinding of outcome assessment and selecting reporting. Full risk of bias assessment is available in Figure 2 and Figure 3.\n\nAs a result of the degree of heterogeneity in terms of study interventions and the incomplete outcome data, it was determined that a meta-analysis of the outcomes was not possible. We carried out a meta-analysis of the complications of pull-out suture versus bone anchor, as two studies had compared these different surgical techniques16,17. The complication rate of pull out suture fixation was higher than that of bone anchor repair (risk ratio, 3.92; 95% CI, 1.07–14.32; P=0.04). Although suggesting a higher rate of complication, this should be interpreted with caution due to the high risk of bias in the included studes, reducing the reliability of the data and subsequent meta-analysis.\n\n\nDiscussion\n\nThe key finding of this systematic review is that is that no study exists comparing non-operative to surgical intervention in the treatment of complete ruptures of the UCL of the thumb. The only studies which have compared interventions are at high risk of bias, particularly in the areas of blinding of outcome assessment and selective outcome reporting. There is weak evidence to suggest that early mobilisation of the thumb MCPJ may be beneficial following surgical repair. There is weak evidence that the pull out suture fixation has a higher rate of adverse events when compared to bone anchor repair.\n\nA systematic review by Samora et al. summarised the outcomes after both non-operative and operative treatment of complete UCL ruptures19. They found that the vast majority of the evidence base was low quality retrospective case series and that only a small minority of patients were treated non-operatively. It was also shown that there was no significant difference in outcome between repair of acute injury and reconstruction after chronic injury.\n\nLandsman et al. demonstrated generally good results when managing complete ruptures with splintage with only 15% failing this regime non operative treatment4; notably, 30% of the patients in this series had displaced fractures and all patients had more than 30° laxity in 30° of MCPJ flexion. A case series reported by Pichora et al. also demonstrated generally satisfactory functional results with functional bracing, even in the 5 patients who were judged to have sustained true Stener lesions20; notably, the three patients who failed functional bracing could not be predicted by the initial clinical tests. Case series purely relating to avulsion fractures of the UCL have shown contrasting results. For example Kuz et al.21 demonstrated satisfactory outcomes in all patients but a non union rate of 25%, this contrasts with the results of Dinowitz et al.22, which demonstrated poor functional results in patients treated non-operatively for minimally displaced fractures.\n\nThere is a widely varying rate of Stener lesions in the literature, it being as low as 12% in the series by Pichora et al.20 and as high as 70% in other series21. The reasons underlying the variability in the rate of the Stener lesion are likely multiple and complex. One aspect of this conundrum appears to be the clear problems with the reliability and accuracy of the radiological diagnosis of the Stener lesion, particularly relating to MRI3. Although there are some high quality studies describing the reliability of ultrasound, there are no high quality studies relating to MRI3. Mahajan et al. demonstrated excellent agreement between radiologists in determining whether the UCL had completely ruptured; however, the presence or absence of a Stener lesion was not assessed radiologically6. Milner et al. have recently argued that any displacement of greater than 3 mm (grade 3 by their system) should be treated operatively, owing to the observed high chance that these patients will fail with non operative treatment9.\n\nThe recent study by Stoop et al. assessed 383 UCL injuries treated at three different hospitals in a singe American city11. In total, 30% of cases were avulsion fractures and 11% of cases were investigated with an MRI scan. Certain patient characteristics were associated with a higher rate of operative intervention, for example greater age and more displaced fractures. However some factors which were unrelated to patient characteristics were predictive of operative intervention, such as having an MRI and being treated by certain surgeons. It was felt that because the preoperative diagnosis of a Stener lesion has limited reliability and accuracy, the rates of surgery may vary based on surgeon beliefs, preferences and values.They also stated that ”some surgeons believe some non-Stener injuries benefit from operative treatment”11.\n\nThis review has demonstrated that all six studies of an intervention with a comparator in UCL treatment are at high risk of bias. The blinding of participants would clearly not be possible in a trial of surgery versus non operative treatment; however, it is a recurrent theme that outcomes were assessed by non-blinded assessors (often the treating surgeon), which significantly increases the chance that detection bias will influence patient outcomes. None of the RCTs published a trial protocol with a specified primary outcome, while only the study by Rocchi et al.14 used validated patient-reported outcome measures (Dreiser index and VAS). There was also a failure to adequately report all outcomes, with only one study reporting adequate data for all outcomes to allow further analysis. None of the three RCTs included a power calculation. While the retrospective nature of the comparative case series introduces several potential sources of bias which may have influenced these results.\n\n\nConclusions\n\nThere is no prospective randomised or observational evidence to support operative intervention compared to non-operative treatment for acute complete ruptures of the ulnar collateral ligament of the thumb. There is weak evidence to suggest that early mobilisation may be beneficial following surgical repair. Further research is necessary in order to better define which patients benefit from which specific interventions.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nJ.C.R.W. is funded by the NIHR as an academic clinical fellow.\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. PRISMA checklist.\n\nClick here to access the data.\n\nSupplementary File 2. Full search histories.\n\nClick here to access the data.\n\nSupplementary File 3. Forest plot of Crowley et al.13 risk ratio of adverse events.\n\nClick here to access the data.\n\nSupplementary File 4. Forest plot of Katolik et al.16 risk ratio of adverse events.\n\nClick here to access the data.\n\nSupplementary File 5. Forest plot of Lane et al.17 risk ratio of adverse events.\n\nClick here to access the data.\n\nSupplementary File 6. Forest plot of Lane et al.17 risk ratio of full versus partial ROM.\n\nClick here to access the data.\n\nSupplementary File 7. Forest plot of Lane et al.17 risk ration of overall outcome full versus partial.\n\nClick here to access the data.\n\nSupplementary File 8. Forest plot of Lane et al.17 strength recovery full versus partial risk ratio.\n\nClick here to access the data.\n\nSupplementary File 9. Forest plot of pull out suture versus anchor complication risk ratio.\n\nClick here to access the data.\n\nSupplementary File 10. Forest plot of Rocchi et al.14 risk ratio of adverse events.\n\nClick here to access the data.\n\nSupplementary File 11. Forest plot of Rocchi et al.14 Dreiser index.\n\nClick here to access the data.\n\nSupplementary File 12. Forest plot of Rocchi et al.14 Range of movement.\n\nClick here to access the data.\n\nSupplementary File 13. Forest plot of Rocchi et al.14 VAS.\n\nClick here to access the data.\n\nSupplementary File 14. Forest plot of Rocchi et al.14 pinch strength.\n\nClick here to access the data.\n\nSupplementary File 15. Forest plot of Saetta et al.18 risk ratio of adverse events.\n\nClick here to access the data.\n\nSupplementary File 16. RevMan 5 file containing the full data extracted from the studies.\n\nClick here to access the data.\n\n\nReferences\n\nPulos N, Shin AY: Treatment of Ulnar Collateral Ligament Injuries of the Thumb: A Critical Analysis Review. JBJS Rev. 2017; 5(2): pii: 01874474-201702000-00005. PubMed Abstract \| Publisher Full Text\n\nCarlsen BT, Moran SL: Thumb trauma: Bennett fractures, Rolando fractures, and ulnar collateral ligament injuries. J Hand Surg Am. 2009; 34(5): 945–952. PubMed Abstract \| Publisher Full Text\n\nPapandrea RF, Fowler T: Injury at the thumb UCL: is there a Stener lesion? J Hand Surg Am. 2008; 33(10): 1882–1884. PubMed Abstract \| Publisher Full Text\n\nHaramati N, Hiller N, Dowdle J, et al.: MRI of the Stener lesion. Skeletal Radiol. 1995; 24(7): 515–518. PubMed Abstract \| Publisher Full Text\n\nRhee PC, Jones DB, Kakar S: Management of thumb metacarpophalangeal ulnar collateral ligament injuries. J Bone Joint Surg Am. 2012; 94(21): 2005–2012. PubMed Abstract \| Publisher Full Text\n\nMahajan M, Tolman C, Würth B, et al.: Clinical evaluation vs magnetic resonance imaging of the skier's thumb: A prospective cohort of 30 patients. Eur J Radiol. 2016; 85(10): 1750–1756. PubMed Abstract \| Publisher Full Text\n\nAbrahamsson SO, Sollerman C, Lundborg G, et al.: Diagnosis of displaced ulnar collateral ligament of the metacarpophalangeal joint of the thumb. J Hand Surg Am. 1990; 15(3): 457–460. PubMed Abstract \| Publisher Full Text\n\nJones MH, England SJ, Muwanga CL, et al.: The use of ultrasound in the diagnosis of injuries of the ulnar collateral ligament of the thumb. J Hand Surg Br. 2000; 25(1): 29–32. PubMed Abstract \| Publisher Full Text\n\nMilner CS, Manon-Matos Y, Thirkannad SM: Gamekeeper's thumb--a treatment-oriented magnetic resonance imaging classification. J Hand Surg Am. 2015; 40(1): 90–95. PubMed Abstract \| Publisher Full Text\n\nHergan K, Mittler C, Oser W: Ulnar collateral ligament: differentiation of displaced and nondisplaced tears with US and MR imaging. Radiology. 1995; 194(1): 65–71. PubMed Abstract \| Publisher Full Text\n\nStoop N, Teunis T, Ring D, et al.: Variation in the Rate of Surgery for Ulnar Collateral Ligament Injury of the Metacarpophalangeal Joint of the Thumb. Hand (N Y). 2017; 12(5): 512–517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggins JPT, Green S: Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 [updated March 2011]. The Cochrane Collaboration, 2011. 2011. Reference Source\n\nCrowley TP, Stevenson S, Taghizadeh R, et al.: Early active mobilization following UCL repair With Mitek bone anchor. Tech Hand Up Extrem Surg. 2013; 17(3): 124–127. PubMed Abstract\n\nRocchi L, Merolli A, Morini A, et al.: A modified spica-splint in postoperative early-motion management of skier's thumb lesion: a randomized clinical trial. Eur J Phys Rehabil Med. 2014; 50(1): 49–57. PubMed Abstract\n\nSollerman C, Abrahamsson SO, Lundborg G, et al.: Functional splinting versus plaster cast for ruptures of the ulnar collateral ligament of the thumb. A prospective randomized study of 63 cases. Acta Orthop Scand. 1991; 62(6): 524–526. PubMed Abstract \| Publisher Full Text\n\nKatolik LI, Friedrich J, Trumble TE: Repair of acute ulnar collateral ligament injuries of the thumb metacarpophalangeal joint: a retrospective comparison of pull-out sutures and bone anchor techniques. Plast Reconstr Surg. 2008; 122(5): 1451–1456. PubMed Abstract \| Publisher Full Text\n\nLane LB: Acute Grade III ulnar collateral ligament ruptures. A new surgical and rehabilitation protocol. Am J Sports Med. 1991; 19(3): 234–238. PubMed Abstract \| Publisher Full Text\n\nSaetta JP, Phair IC, Quinton DN: Ulnar collateral ligament repair of the metacarpo-phalangeal joint of the thumb: a study comparing two methods of repair. J Hand Surg Br. 1992; 17(2): 160–163. PubMed Abstract \| Publisher Full Text\n\nSamora JB, Harris JD, Griesser MJ, et al.: Outcomes after injury to the thumb ulnar collateral ligament--a systematic review. Clin J Sport Med. 2013; 23(4): 247–254. PubMed Abstract \| Publisher Full Text\n\nPichora DR, McMurtry RY, Bell MJ: Gamekeepers thumb: a prospective study of functional bracing. J Hand Surg Am. 1989; 14(3): 567–573. PubMed Abstract \| Publisher Full Text\n\nLouis DS, Huebner JJ Jr, Hankin FM: Rupture and displacement of the ulnar collateral ligament of the metacarpophalangeal joint of the thumb. Preoperative diagnosis. J Bone Joint Surg Am. 1986; 68(9): 1320–1326. PubMed Abstract \| Publisher Full Text\n\nDinowitz M, Trumble T, Hanel D, et al.: Failure of cast immobilization for thumb ulnar collateral ligament avulsion fractures. J Hand Surg Am. 1997; 22(6): 1057–63. PubMed Abstract \| Publisher Full Text" }	[ { "id": "34855", "date": "21 Jun 2018", "name": "Harpal Uppal", "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 interesting article systematically reviews the literature base regarding injuries of the thumb ulnar collateral ligament. A large number of studies (158) were read and assessed to identify 6 eligible comparative studies. The 6 studies in question appear to be highly heterogeneous and have a high risk of being susceptible to bias. The quality of published data is too poor for the study to achieve its initial goal of performing a meta analysis. This is in itself a valuable piece of information which can be used to help drive further research. The search strategy is well described and repeatable and conducted according to the PRISMA checklist. Overall this is a well written paper, of publishable standard, describing poor quality data which is of particular value to researchers planning new studies investigating or designing new studies concerning the ulnar collateral ligament.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "34857", "date": "02 Jul 2018", "name": "Charles Pailthorpe", "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 aim was to perform a systematic review of the effectiveness of available interventions for acute complete rupture of the ulnar collateral ligament of the thumb in terms of patient-reported outcome measures and to assess the rates of adverse outcomes associated with these interventions.\nTheir methodology appears overall sound, however I have some concerns over their request for additional data from the authors of the selected papers. If this data was not included in the original papers how can it be accepted retrospectively. Overall the authors have achieved their aim of the systematic review however they have added a large amount of extra data particularly concerning diagnosis (MRI and US).\nIn general I think the paper has merit to be published but in their stated aim the authors should include that they reviewed the literature concerning diagnosis as well.\n11th June 2018: The status of this report has been updated from ‘Approved with reservations’ to ‘Approved’ in response to the author comments.\n\nI thank the authors for their comments and accept that no additional data was either sourced or utilised. Also, I accept their comments on the diagnostic component in the article and accept that on balance it is useful in its contribution to the overall aim.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "3790", "date": "03 Jul 2018", "name": "Benjamin Dean", "role": "Author Response", "response": "Many thanks for your comments which we have attempted to address below.“Their methodology appears overall sound, however I have some concerns over their request for additional data from the authors of the selected papers. If this data was not included in the original papers how can it be accepted retrospectively.”In terms of requesting additional data, we used the approach described and recommended by the Cochrane group1. No authors were able to provide any additional data, as summarised in Table 2 of our study, therefore no additional data was accepted retrospectively.“Overall the authors have achieved their aim of the systematic review however they have added a large amount of extra data particularly concerning diagnosis (MRI and US).”We have described some findings relating to diagnosis within the introduction and discussion with the aim of providing context and clinical relevance to the systematic review. Obviously, this element has not been performed systematically and this was not our intention. If the reviewer feels the context as regards diagnosis could be better summarised then we are happy to consider any suggestions which may augment the discussion. Should the reviewer feel that the statements regarding radiological investigations are superfluous, then we would be happy to consider excluding them. 1. Young T, Hopewell S. Methods for obtaining unpublished data. Cochrane database of systematic reviews (Online). Nov 9 2011(11):MR000027." } ] } ]	1	https://f1000research.com/articles/7-714
https://f1000research.com/articles/7-319/v1	14 Mar 18	{ "type": "Software Tool Article", "title": "segment_liftover : a Python tool to convert segments between genome assemblies", "authors": [ "Bo Gao", "Qingyao Huang", "Michael Baudis", "Bo Gao", "Qingyao Huang" ], "abstract": "The process of assembling a species’ reference genome may be performed in a number of iterations, with subsequent genome assemblies differing in the coordinates of mapped elements. The conversion of genome coordinates between different assemblies is required for many integrative and comparative studies. While currently a number of bioinformatics tools are available to accomplish this task, most of them are tailored towards the conversion of single genome coordinates. When converting the boundary positions of segments spanning larger genome regions, segments may be mapped into smaller sub-segments if the original segment’s continuity is disrupted in the target assembly. Such a conversion may lead to a relevant degree of data loss in some circumstances such as copy number variation (CNV) analysis, where the quantitative representation of a genomic region takes precedence over base-specific accuracy. segment_liftover aims at continuity-preserving remapping of genome segments between assemblies and provides features such as approximate locus conversion, automated batch processing and comprehensive logging to facilitate processing of datasets containing large numbers of structural genome variation data.", "keywords": [ "Genome assembly", "liftover", "remap", "copy number segment." ], "content": "Introduction\n\nThe first draft version of human genome was published in 20011. In subsequent years, several new editions were released to perfect the quality of the genome assembly. The current version of human genome (GRCh38, UCSC hg38) was made available in 2013, with the latest revision (Grch38.p12) still containing more than 10 million unplaced bases (see NCBI website). Over the years, large numbers of genomic studies have been performed, generating data mapped to different versions of the reference genome. However, when performing genome analyses integrating data from multiple resources, it is imperative to convert all data to the same genomic coordinate system.\n\nTwo general methodologies are used for conversion between coordinates from different genome assemblies. The first approach is to re-align the original sequence data to the target assembly. This method could provide the best result but is very time consuming, and is not possible when the original sequence data is not available or does not consist of direct sequences (i.e. segmentation of array based data). Another approach is to convert the coordinates of genome data between assemblies by using a mapping file. This method, although bearing a side effect of minor information loss, for most applications provides a good balance between performance and accuracy.\n\nCurrently, three tools are in widespread use for the conversion between genome assemblies by coordinates: liftOver from University of California, Santa Cruz (referred as UCSC liftOver in the following article)2; CrossMap from Zhao3; and Remap from NCBI4. The UCSC liftOver tool exists in two flavours, both as web service and command line utility. It offers the most comprehensive selection of assemblies for different organisms with the capability to convert between many of them. CrossMap has the unique functionality to convert files in BAM/SAM or BigWig format. It generates almost identical results as UCSC liftOver, but is not optimised for converting genome coordinates between species. Remap provides for each organism a comprehensive list of major assemblies and the corresponding sub-versions. It can also perform cross species mapping, however, with only a limited number of organisms.\n\nAll those tools are efficient in coordinate conversion and provide almost identical results. However, as shown in Figure 1a, challenges arise when dealing with genome segments that are not continuous anymore in the target assembly; there, these three tools take on different strategies. CrossMap and UCSC liftOver break the segment into smaller segments and map them to different locations. Remap keeps the integrity of the segment and maps the span to the target assembly.\n\n(a) When lifting a segment to another assembly, the landscape of the segment may be affected by indels and copy number variations, but the overall span of the segment does not change significantly. (b) When the end positions cannot be converted by the UCSC liftOver, the nearby regions will be searched for convertible positions as approximation. (c) Quality control checks for changes of chromosome or size to make sure the segment is converted properly.\n\nIn research such analysis of copy number variation (CNV) data, where the quantitative representation of a genomic range takes precedence over base-specific representation, the integrity of a continuous segment indicates the proper conversion between assemblies, but may not be a direct outcome of current re-mapping approaches. Although Remap can convert contiguous segments, it only provides web service with submission limits, which is difficult to use for large scale or pipelined applications. The limitation to single input files is a general limitation of those tools, which precludes their direct use in comparative studies which may require to work with tensor hundreds of thousands of files, as indicated through our own projects and requests from the research community.\n\nIn this article,we introduce segment_liftover, a tool to perform an integrity-preserving conversion of genomic segments data between genome assemblies. It features two major functional additions over existing tools: First, re-conversion by locus approximation, in instances where a precise conversion of genomic positions fails; and second, the capability to handle any number of files and optional integration into data processing pipelines with supporting features such as automatic file traversal, interruption resumption and detailed logging.\n\n\nMethods\n\nsegment_liftover can convert both probe files and segment files at the same time or in separate runs. It starts from a structured directory or a list of files, then traverses and converts all files meeting the specified name pattern, and finally outputs to a designated directory. To convert a probe file, segment_liftover will first use the UCSC liftOver to convert the file, then apply an approximate conversion on probes that the UCSC liftOver failed to convert. To convert a segment file, segment_liftover will use the UCSC liftOver to convert the start and end positions of segments. A successful segment conversion needs to satisfy the following four criteria:\n\npositionnew_start ≠ ∅ (1)\n\npositionnew_end ≠ ∅ (2)\n\nchromosomenew_start = chromosomenew_end (3)\n\n\n\nWhere β controls the threshold of the length ratio and the default value is set to 2. If criteria (1) or (2) fails, segment_liftover will apply an approximate conversion; if the conversion still fails, it is reported as unconvertible. If criteria (3) or (4) fails, the conversion is reported as rejected (Figure 1c). The reason of failure is recorded in log files.\n\nWhen a position cannot be converted by the UCSC liftOver, segment_liftover will attempt an approximate conversion and try to find a convertible position in the adjacency (Figure 1b). The range and the resolution of the search is defined by parameters –range and –step_size, respectively.\n\nThe segment_liftover tool is implemented in Python. The package is available for both Linux and OSX. It requires a minimum of 2G memory and the capacity of running Python 3. We recommend an installation using pip in a Python virtual environment. segment_liftover requires and depends on the UCSC liftOver program. A chain file, which provides alignments from source to target assembly, is also required. The chain files between common human assemblies (hg18, hg19 and hg38) are included in the program package. Chain files of other species and assemblies are available from the USCS Genome Browser. Figure 2 illustrates the work-flow of segment_liftover.\n\n(1) It can take either a folder or a file containing the list of files as the input. (2) It will try to convert by approximation when UCSC liftOver fails to convert a coordinate. (3) The directory structure will be kept in the output folder and detailed log files are also available.\n\n\nUse cases\n\nIn this section, we provide two examples of using segment_liftover to convert probes and segments, respectively. The two examples are part of the pipeline which updates the arrayMap database, a reference resource of somatic genome copy number variations in cancer5, from human genome assembly hg19 to hg38. In the first example, we converted 44,632 probe files and 44,471 segment files from hg19 to hg38. The probe data were generated from nine Affymetrix genotyping array platforms, which currently only support annotations for hg19. Circular binary segmentation (CBS) analysis (DNAcopy R-package) was used to infer copy number segments from log2 values of probes. The final segment files contain a list of genomic regions separated by their copy number values6. We ran the segment_liftover tool on a 12-core, 128GB RAM machine with 8 parallel processes. It took 42 hours to convert 44,632 probe files with 5.5 billion probe positions and 40 minutes to convert 44,471 segment files with 4.8 million segments.\n\nOverall, more than 99.99% of probes and more than 99% of segments could be directly converted from hg19 to hg38 (Figure 3). As conversion of segments is more complicated and involves a quality control procedure to ensure the meaningfulness of the segment, it is expected to have a higher number of unconvertible segments than probes. As shown in Figure 4, the unconvertible regions are mainly around telomeres, centromeres, or other gene-sparse locations. In total, 38 genetic elements were found to be affected by the conversion (description in Supplementary Table 1).\n\nDirectly converted is the sum of successful conversion from UCSC liftOver; approximately converted is the sum of successful re-conversion in locus adjacency; converted but rejected is the sum of all rejections from quality control; unconvertible is the sum of everything that is not converted.\n\nUnconvertible positions are marked red on the karyogram, annotated with HGNC symbol or ENSEMBL gene ID (if HGNC not available), retrieved from biomaRt_2.30.0.\n\nIn the second example, we compared the performance of different conversion strategies using 1,000 samples randomly drawn from the first example (Table 1). Copy number segments were generated using four different strategies (Figure 5): (1) segments in hg19 were generated from probes in hg19 using the aforementioned standard pipeline; (2) segments in hg38 were generated with probes converted from hg19 to hg38 using the standard pipeline; (3) segments converted from hg19 to hg38 with approximate conversion; (4) segments converted from hg19 to hg38 without approximate conversion.\n\nTable 2 shows the comparison of segments conversion in average between (3), (4) and (2) (complete table in Supplementary Table 2). Exact segment matches are categorized as \"perfect\"; \"minor difference\" is defined the same as condition (3) of quality control; the rest of the segments are categorized as \"significant difference\"; \"sum\" is the total number of segments of a sample in average. By comparing the sum of reference hg19, reference hg38 and approximation, it shows that the conversion result is very close to the result of the standard pipeline. The difference between converted and generated sums is much smaller than the difference between two generated sums of different genome versions. On average, the approximate conversion could rescue one additional segment per file. Finally, we zoom into a specific example on chromosome 9 in GSM276858 (illustrated in Figure 5). Because of the removal of probes from hg19 to hg38, the second segment will be lost without approximate conversion.\n\nSegments directly processed from hg38 probes are used as reference (top right). hg19 segments converted with approximation (middle right) and without approximation (bottom right) are used for comparison.\n\nThe two examples above demonstrated the efficiency and effectiveness of segment_liftover in processing large number of probe and segment files. It can provide conversion results that are similar to results generated from the standard pipeline. Moreover, with approximate conversion, the number of properly converted segments is slightly increased. In general, segment_liftover is able to provide reliable conversions and the ease of use.\n\n\nSummary\n\nTranslation between genome versions of sequencing data is a tedious but crucial task in bioinformatics. With the functionalities of automated batching, approximate conversion and segment conversion, segment_liftover can dramatically reduce the complexity and workload of such data processing. Furthermore, segment_liftover’s detailed logs of execution result provide an easy and clear foundation for follow up analysis.\n\n\nSoftware availability\n\n1. pip version: https://pypi.python.org/pypi/segment-liftover\n\n2. Latest source code: https://github.com/baudisgroup/segment-liftover\n\n3. Archived source code as at time of publication: https://dx.doi.org/10.5281/zenodo.11868037\n\n4. Software license: MIT", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nBG is recipient of a grant from the China Scholarship Council (CSC 201606620032). QH is supported through the University of Zurich’s \"CanDoc\" program.\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 Paula Carrio Cordo and the participants of the \"Zurich Seminars in Bioinformatics\" for helpful discussions.\n\n\nSupplementary material\n\nSupplementary Table 1 : genetic elements affected by the conversion The 38 genetic elements that are affected by the segment_liftover conversion from hg19 to hg38.\n\nClick here to access the data.\n\nSupplementary Table 2 : complete conversion results of different strategies A complete list of conversion results of comparing different conversion strategies using 1000 random samples.\n\nClick here to access the data.\n\n\nReferences\n\nKent WJ, Haussler D: Assembly of the working draft of the human genome with GigAssembler. Genome Res. 2001; 11(9): 1541–1548. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuhn RM, Haussler D, Kent WJ: The ucsc genome browser and associated tools. Brief Bioinform. 2013; 14(2): 144–161. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao H, Sun Z, Wang J, et al.: Crossmap: a versatile tool for coordinate conversion between genome assemblies. Bioinformatics. 2014; 30(7): 1006–1007. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNCBI Resource Coordinators: Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2016; 44(D1): D7–D19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCai H, Gupta S, Rath P, et al.: arraymap 2014: an updated cancer genome resource. Nucleic Acids Res. 2015; 43(Database issue): D825–D830. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlshen AB, Venkatraman ES, Lucito R, et al.: Circular binary segmentation for the analysis of array-based DNA copy number data. Biostatistics. 2004; 5(4): 557–572. PubMed Abstract \| Publisher Full Text\n\nGao B, Baudis M: baudisgroup/segment-liftover: First public version (Version 0.948). Zenodo. 2018. Data Source" }	[ { "id": "31947", "date": "26 Mar 2018", "name": "Oscar Krijgsman", "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 discusses a new tool developed by the authors that improves the ‘liftover’ between genomic builds. “Segment_liftover” is built on the already available liftover tool from UCSC with the novelty that it the tool implements a better conversion between genomic builds for stretches of the genome (segments) like in for example DNA copy number analyses. In addition, the tool provides options to perform the analysis in batches and provides log files of execution and results. The tool, as available on github, provides ample documentation and examples to run without experiencing too many difficulties.\nThe authors provide an interesting new tool that shows an improvement over existing tools. In general, the manuscript is clear and concise. However, a few minor points need to be addressed to improve the interpretation of the results described in the manuscript.\n\nThe second use case is basically a direct comparison between UCSC liftover and the newly developed segment_liftover tool. However, this is not explicitly mentioned in the text. In the current version it is not evident that you did the nice/required comparison with a currently available tool. Furthermore, a little more explanation on the 4 different strategies would benefit the reader.\n\nFigure 3 does not convincingly convey its message. For example, it is not clear from the figure that the percentage of unconvertible segments is higher that probes although properly mentioned in the text. But also, the difference between the first bars is over a factor 1000. I understand the choice of a log10 scale but the authors might consider a different way of showing this data (table? Add percentages or number?).\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": "3694", "date": "08 Jun 2018", "name": "Bo Gao", "role": "Reader Comment", "response": "\" The second use case is basically a direct comparison...\"Our intention is to compare the performance of segment_liftover with the standard copy number segment calling procedure, which generates segments from probes. We have expanded and revised this part to provide more information about the settings and the purpose.\" Figure 3 does not convincingly convey its message...\"We also had discussions about the best way to show the information, now we have replaced the figure with a table for clarity. Thank you for the suggestions!" } ] }, { "id": "32847", "date": "17 Apr 2018", "name": "Ryan K. Dale", "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 how using existing genome coordinate conversion tools can be problematic on large domains in which continuity of the converted region is preferred over precision of the coordinate conversion. They describe a method that iteratively retries the conversion and applies QC criteria to the original and iterative conversions. The method is implemented using a Python package that wraps the UCSC liftover tool and performs the iterative remapping and QC steps, and provides this functionality over directories of files instead of on a per-file basis.\nTo make it scientifically sound there should be a clear example in the documentation that works (details below). I have additional concerns and suggestions, particularly about the code, that I hope will improve the paper and the tool.\nMajor comments:\n- Figure 3 is difficult to interpret as-is because some probes are double-counted across bars. I think a better approach would be to have a stacked bar for which the full height indicates the total number of probes that can be subset into converted and not-converted. These can be further subset by colors or by an adjacent stacked bar to indicate the proportion of converted that were directly converted or approximately converted, and the proportion of QC-failed.\n- I see in the code that there are some human-specific hard-coded conversions between chr23/24 and chrX/Y. This may cause unexpected results when running on other genomes. One solution would be to run a check on the chromosomes observed in the chainfile to see if it looks like a human liftover before doing the 23/24 -> X/Y conversions.\n- Currently the liftOver executable is required to be in the working directory or manually specified with the \"--liftover\" arg. A friendlier solution would be to expect liftOver to be found on the PATH variable (while also keeping the ability to explicitly specify if needed). This avoids having to symlink the executable to the working directory or requiring system-dependent command-line arguments.\n- A \"logs\" directory is created in the working directory, even just after running \"segment_liftover --help\". It would be nice to defer creating this directory until it's actually needed. This also means that running parallel jobs on a cluster from the same directory will clobber the log files, making debugging difficult. It would be better to expose log directory configuration on the CLI and/or put the logs directory as a subdirectory of the output directory by default.\n- the \"tmp\" directory behaves the same way (clutters the working directory; clobbers files when running in parallel). Furthermore it's not cleaned up after running. Better would be to use Python's tempfile module, which respects the environment's TMPDIR variable and is periodically automatically cleaned.\n- Example data in the correct directory structure should be provided, and a single command to run that data should be provided. That way the user can easily verify that everything is installed correctly. As it is, I had to create the directory structure and paste the example segments content from the README into a new file in that directory. That allowed segment_liftover to run, but then it failed on the example segments data in the README. Digging around in the logs, in general.log there appears to be an issue with int vs str expectations when loading as pandas.DataFrame (this is with pandas v0.22.0).\n- Are there requirements for chromosome names in segment files or probe files? The examples use integer chromosome names, but liftOver chainfiles use UCSC \"chr\" chromosome names. It appears that using an input file with \"chr\" names fails, again from expectations about int vs str columns in pandas. It would be good to describe what kind of chromosome names are supported (or link to a segments file specification if there is one).\nMinor comment: Typo in text, \"tensor\" -> \"tens or\", and various typos in help text when running segment_liftover\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": "3693", "date": "08 Jun 2018", "name": "Bo Gao", "role": "Reader Comment", "response": "\"- Figure 3 is difficult to interpret...\"We also discussed the best way to show this. Now it's replaced with a table. \"- I see in the code that there are some human-specific hard-coded conversions...\"Sorry for this overlook, the tool was originally developed for the human genome, and we forgot to modify this, now the hard coding replaced with generally coding. Following your recommendation, we investigated the feasibility of inferring valid chromosome names from the chainfile, but it turns out for some small genomes, the chainfile may not contain all valid chromosome names. In the end, as UCSC liftOver will ignore unrecognized chromosome names, we decide to pass the duty of providing correct chromosome names to the user.\" - Currently the liftOver executable is required to be in the working directory...\"Now the tool looks for the liftOver in the system path by default, and users can still manually specify with \"-l\". \" - A \"logs\" directory is created in the working directory...\"Modifications: (1) the log file and directory is created only after something is written to the file. (2) the logs/ directory is moved to the output directory by default. (3) users can also specify the log directory with a new option.\" - the \"tmp\" directory behaves the same way...\"The tmp/ directory is also moved to the output directory and will be properly removed after execution. Because sometimes we need to inspect the temp files, we decide not to use the Python module for easy accessing. \" - Example data in the correct directory structure should be provided...\"A new option \"--demo\" is added to provide a small demo dataset and a quick run.\" - Are there requirements for chromosome names...\"README and MANUAL have been updated to provide a better explanation of accepted format. \"Typo in text...\"All text has been revised.Thank you very much for all the valuable suggestions!" }, { "c_id": "3718", "date": "15 Jun 2018", "name": "Michael Baudis", "role": "Author Response", "response": "Thank you for this very constructive referee report! We hope that the implemented changes satisfy the your wishes for improvements." } ] } ]	1	https://f1000research.com/articles/7-319
https://f1000research.com/articles/7-711/v1	07 Jun 18	{ "type": "Research Article", "title": "Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse", "authors": [ "Suzanne O. Nolan", "Joaquin N. Lugo", "Suzanne O. Nolan" ], "abstract": "Background: Loss of FMR1 is associated with Fragile X syndrome, amongst the most prevalent inherited intellectual disability. Despite extensive research in this area, previous studies have failed to detect consistent evidence of cognitive impairments in the Morris water maze (MWM) task in the Fmr1 knockout (KO) mouse. However, few studies have examined cognitive flexibility in a reversal form of the MWM task, which may illuminate subtle learning deficits. Methods: Adult male Fmr1 wildtype (WT) and KO mice were bred and tested in the MWM reversal paradigm. The testing paradigm consisted of two blocks per day, with 4 trials per block to locate a hidden platform. After the last trials on the fourth day of testing, the animals were given a probe trial with the platform removed. The following week, the location of the platform was switched to the opposite quadrant and the animals received 2 more days of testing, with 4 blocks in total. Results: As expected, Fmr1 KO mice did not display a learning deficit during the acquisition phase, Fgenotype (1, 24) = 0.034, p = 0.854, and performed similarly on the probe trial, Fgenotype (1, 23) = 0.024, p = 0.877. However, during the reversal phase of learning, Fmr1 KO mice showed deficits in their ability to learn the new location of the platform, Fgenotype (1, 23) = 3.93, p = 0.059. Further independent samples t-testing revealed that KO animals displayed significantly higher latency to reach the hidden platform during the third trial, t(23) = -2.96, p < 0.01. Conclusions: While previous studies have not demonstrated deficits in spatial memory in the Fmr1 KO model, it is possible that the acquisition phase of the task is less sensitive to deficits in learning. Future studies using this model to evaluate therapeutic interventions should consider utilizing the MWM reversal paradigm.", "keywords": [ "Cognitive flexibility", "Autism", "Morris water maze", "Prefrontal cortex", "FMRP" ], "content": "Introduction\n\nFragile X syndrome (FXS) is a neurodevelopmental disorder, caused by a trinucleotide expansion mutation in the FMR1 gene, and is also one of the most prevalent inherited forms of intellectual disability1. FXS is often modeled using the Fmr1 knockout (KO) mouse, which can be characterized by several behavioral phenotypes, including alterations in sociability and deficits in fear memory2–4. Aside from deficits in spatial and non-spatial learning, one understudied facet of intellectual disability is the ability to incorporate new information into existing learning, termed cognitive flexibility. Cognitive flexibility can be studied in rodents using a variant of the Morris water maze (MWM) paradigm5,6. In the MWM reversal paradigm, the location of the hidden platform is moved, and the latency to adjust to the new location is measured. As expected, several reports find evidence of impairments in reversal learning in the Fmr1 KO mouse across multiple strains, the C57BL/6J backcrossed strain7,8 and the albino C57BL/6J background2. However, other studies have been unable to replicate these findings, and further investigation points to the possibility of background strain differences9. Previous reports have not detected any impairments in reversal learning in the FVB.129 strain10. However, it may be that this paradigm is perhaps even more sensitive to methodological differences. The current study adds to this literature by using the FVB.129 strain in a previously utilized paradigm.\n\n\nMethods\n\nMale Fmr1+/+ and female Fmr1+/- FVB.129P2-Pde6b+Tyrc-ch Fmr1tm1Cgr/J (Stock No: 004624, The Jackson Laboratory, Bar Harbor, ME, USA) mice were used as breeders (9 total breeding pairs) to produce the following groups: male WT and male KO pups. Breeding pairs were of the following groupings: WT Female/WT Male (n = 2), KO Female/KO Male (n = 5), WT Female/KO Male (n = 2). Genotype was determined from toe clippings taken prior to age postnatal day (PD) 12 (Mouse Genotype, Escondido, CA, USA). The final sample sizes were as follows: nmale WT = 10, nmale KO = 16. Target samples sizes (n = 10) were calculated a priori using a power calculation in G*Power 3.1 with the following parameters: f = 0.50 (large effect), α = 0.05, power (1 – β) = 0.80, for the F family of tests with two groups and 8 repeated measures (trials). All pups were housed in individual cages (Allentown Caging PC7115HT, Allentown, PA, USA), filled with sani-chip bedding (7090 Teklad, Envigo, Somerset, NJ, USA). Prior to weaning on PD21, pups were housed with parents (1 male and 2 females) and littermates (up to 12 pups). Following weaning, subjects were housed with mixed genotype littermates, no more than 5 to a cage. The light cycle was kept at 12 hr. light, and the colony room was kept at an ambient temperature of 22° C. Animals had ad libitum access to food and water. All procedures performed were in accordance with Baylor University Institutional Animal Care and Use Committee (Animal Assurance Number A3948-01), as well as the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All efforts were made to ameliorate any stress and harm to the animals, specifically by habituating animals to the testing apparatus and room prior to trial recordings.\n\nAll behavioral testing was conducted during the light cycle, specifically between 8 am and 5 pm. The methods for the current study were adapted as closely as possible from earlier studies of this behavior in the Fmr1 KO mouse (represented in Figure 1)9. Briefly, a 1.3 m diameter white pool was filled with water and made opaque through the addition of non-toxic white paint (Item LT3010, S&S Worldwide, Connecticut). The hidden platform measured 14.5 cm × 14.5 cm and was submerged approximately 2 cm below the water level. The testing paradigm consisted of two blocks per day for 4 days, with 4 trials in each block, for each mouse to test the ability to locate a hidden platform. The mice were habituated to the testing room in their holding cages for 30 minutes prior to the onset of testing. The amount of time spent in each quadrant for each trial was recorded with a ceiling-mounted video camera (Ganz YCH-02, Cary, NC, USA), and analyzed using automated tracking software (Ethovision XT 6, Noldus, Wageningen, Netherlands). After the last trial on the fourth day of testing was completed, the animals were given a probe trial. The probe trial involved removing the platform and allowing the subjects to explore the maze for 60 seconds. During the probe trial, the number of times the animal crossed the location of the hidden platform and the duration of time in each quadrant was calculated. Testing resumed on day 8 after a 3-day rest period. On day 8, the platform was placed in the opposite quadrant from the previous location that housed the hidden platform. Testing progressed as with the initial acquisition phase, with 2 blocks per day for 2 days. On the final day of testing, a visible platform was used to evaluate visual performance as well as swim speed. The visible platform was a two-tiered platform similar to the initial platform, with a second higher tier platform that extended 9.5 cm above the lower platform, allowing the animal to see the platform. The differences in methodology from the cited source were as follows: only four days of acquisition were conducted and the testing paradigm was lengthened to account for a consolidation period between the learning and reversal trials (See Figure 1 for a description of the testing paradigm). One KO animal was excluded from analysis due to a seizure during this task.\n\nA. An overview of the set-up of the testing arena. B. An overview of the progression of testing days.\n\nStatistical analysis was performed in the form of a one-way analysis of variance (ANOVA) with one between-subjects factor (Genotype [wildtype, knockout]) and one within-subjects factor (Trial). All data were analyzed using GraphPad Prism Software 7.0 (San Diego, CA, USA) or IBM SPSS Statistics 23 (Aramonk, NY, USA).\n\n\nResults\n\nTo investigate the effect of genotype on hippocampal spatial memory, animals were tested in the MWM paradigm (Dataset 1). During the 8 blocks of learning trials (Figure 2A), there was a significant within-subjects effect of trial for latency to reach the platform, F(3.56, 85.47) = 30.15, p < .0005. Trial results did not interact significantly with genotype, F(3.56, 85.47) = 1.33, p = 0.24, suggesting both groups learned the location of the platform similarly. Between-subjects analyses indicated no effect of genotype, F(1, 24) = 0.03, p = 0.85. Further independent samples t-testing revealed no differences between WT and KO at any of the 8 different trials, p > 0.05.\n\nFor the probe trial (Dataset 2), as expected, male KO mice demonstrated similar time spent in the target quadrant, F(1,23) = 0.02, p = 0.88 (Figure 2B), compared to male wildtype (WT). Further independent samples t-testing revealed no differences in duration in any of the quadrants, p > 0.05.\n\nA. Fmr1 knockout males show no deficits in acquisition in performance. B. Performance during the probe trial is not impaired in the Fmr1 knockout males. C. When subjected to a reversal learning paradigm, Fmr1 knockout mice display increased latency to the new platform location, demonstrating deficits in cognitive flexibility. Knockout – KO, Wild type - WT.\n\nThe week following the initial learning trials, animals were tested in the reversal learning paradigm (Dataset 3). During the 4 blocks of learning trials (Figure 2C), a one-way ANOVA with repeated measures revealed a significant within-subjects effect of trial, F(3, 69) = 3.8, p < 0.05. Trial results did not interact significantly with genotype, F(3, 69) = 1.28, p = 0.29. However, between-subjects analyses indicated a marginal effect of genotype, F(1, 23) = 3.93, p = 0.059 (Figure 2C). Further independent samples t-testing revealed that KO animals displayed significantly higher latency to reach the hidden platform during the third trial, t(23) = -2.96, p < 0.01. Together, these results demonstrate that Fmr1 KO males demonstrate decreased learning and altogether a lack of cognitive flexibility across all trials of the MWM reversal task.\n\nVisible platform information was also assessed to ensure differences were not due to deficits in vision (Dataset 4). Results were analyzed using a repeated-measures ANOVA across the four visible platform trials on latency to the platform across the two blocks of trials. Results indicated no effect of block, F(1, 24) = 1.341, p = 0.26, nor an interaction of block and genotype, F(1, 24) = 0.0005, p = 0.98. There was not a significant effect of genotype on latency to the platform during these trials either, F(1, 24) = 0.98, p = 0.33 (Figure 3A). Altogether, these data suggest that differences in latency to the platform could not be attributed to deficits in visual perception in the Fmr1 KO male mouse. Moreover, differences in latency to the platform on the previous trials could not be attributed to impairments in swimming abilities, as no differences in swim speed were detected during the visible trials, t(11.17) = 1.526, p = 0.16 (Figure 3B).\n\nA. Fmr1 knockout males showed no deficits in latency to the platform across the two blocks of the visible platform test. B. Fmr1 knockout males showed no deficits in swim speed across the visible platform test. Knockout – KO, Wild type - WT.\n\n\nDiscussion\n\nAs expected, deletion of Fmr1 did not impact initial spatial learning in the MWM. The current study did, however, demonstrate impairments in cognitive flexibility in the Fmr1 KO. As previously mentioned, other studies have not before detected such changes, and this discrepancy could be attributed to methodological differences10. In the aforementioned study, training occurred over 8 days, with only 3 training trials per day, and the reversal paradigm consisted of 4 days, with 3 trials per day. Furthermore, in support of our findings, deficits in long-term potentiation in the prefrontal cortex have been demonstrated in the Fmr1 KO mouse, the area on which the ability to adapt to a new location in this task is dependent on 11–13. Moreover, this ability to adapt to a new location is mediated through multi-synaptic connections between the hippocampus and the prefrontal cortex13. This proposed mechanism further supports our findings of no change to the initial spatial learning phase, as lesions to this area did not impact initial learning performance in spatial navigation11.\n\nThe current study provides preliminary evidence that could be applied to tease apart subtle differences between the male and female Fmr1 KO phenotype, which has been difficult to conclusively evaluate. Future studies should expand upon these findings in females, as many studies have demonstrated a sex-specific effect of loss of Fmr1 on behavior (discussed in a recent review14)2–4,15. Overall, this study corroborates and extends previous evidence of impaired cognitive flexibility in the male Fmr1 KO mouse.\n\n\nData availability\n\nDataset 1– “Learning Trial Data – CSV.csv”\n\nThis datasheet contains the raw data exported from the Ethovision program (Columns A – G) as well as the transformed dataset that was used for analysis for the learning trials (Columns J – T). http://dx.doi.org/10.5256/f1000research.14969.d20606816\n\nDataset 2 – “Probe Trial Data – CSV.csv”\n\nThis datasheet contains the raw data exported from the Ethovision program (Columns A - AB) as well as the transformed dataset that was used for analysis for the probe trial (Columns AC – AI). http://dx.doi.org/10.5256/f1000research.14969.d20606917\n\nDataset 3 – “Reversal Trial Data – CSV.csv”\n\nThis datasheet contains the raw data exported from the Ethovision program (Columns A – G) as well as the transformed dataset that was used for analysis for the reversal trials (Columns J – O). http://dx.doi.org/10.5256/f1000research.14969.d20607618\n\nDataset 4 – “Visible Platform Data – CSV.csv”\n\nThis datasheet contains the raw data exported from the Ethovision program (Columns A – H) as well as the transformed dataset that was used for analysis of the visible platform trials (Columns K – S). http://dx.doi.org/10.5256/f1000research.14969.d20607719", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by funding from the National Institutes of Health, National Institute of Neurological Disorders and Stroke [NS088776].\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 Gregory Smith for his initial training guidance. We would also like to thank Conner Reynolds, Samantha Hodges, Matthew Binder, and Andy Holley for their critical review of the paper.\n\n\nReferences\n\nHagerman RJ, Hagerman PJ: Fragile X syndrome: diagnosis, treatment, and research. 3rd ed. Baltimore: Johns Hopkins University Press. xii. 2002; 540. Reference Source\n\nBaker KB, Wray SP, Ritter R, et al.: Male and female Fmr1 knockout mice on C57 albino background exhibit spatial learning and memory impairments. Genes Brain Behav. 2010; 9(6): 562–74. PubMed Abstract \| Publisher Full Text\n\nDing Q, Sethna F, Wang H: Behavioral analysis of male and female Fmr1 knockout mice on C57BL/6 background. Behav Brain Res. 2014; 271: 72–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNolan SO, Reynolds CD, Smith GD, et al.: Deletion of Fmr1 results in sex-specific changes in behavior. Brain Behav. 2017; 7(10): e00800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorris R: Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods. 1984; 11(1): 47–60. PubMed Abstract \| Publisher Full Text\n\nFmr1 knockout mice: a model to study fragile X mental retardation. The Dutch-Belgian Fragile X Consortium. Cell. 1994; 78(1): 23–33. PubMed Abstract \| Publisher Full Text\n\nKooy RF, D'Hooge R, Reyniers E, et al.: Transgenic mouse model for the fragile X syndrome. Am J Med Genet. 1996; 64(2): 241–5. PubMed Abstract \| Publisher Full Text\n\nD'Hooge R, Nagels G, Franck F, et al.: Mildly impaired water maze performance in male Fmr1 knockout mice. Neuroscience. 1997; 76(2): 367–76. PubMed Abstract \| Publisher Full Text\n\nParadee W, Melikian HE, Rasmussen DL, et al.: Fragile X mouse: strain effects of knockout phenotype and evidence suggesting deficient amygdala function. Neuroscience. 1999; 94(1): 185–92. PubMed Abstract \| Publisher Full Text\n\nLeach PT, Hayes J, Pride M, et al.: Normal Performance of Fmr1 Mice on a Touchscreen Delayed Nonmatching to Position Working Memory Task. eNeuro. 2016; 3(1): pii: ENEURO.0143-15.2016. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartin HGS, Lassalle O, Brown JT, et al.: Age-dependent long-term potentiation deficits in the prefrontal cortex of the Fmr1 knockout mouse model of fragile X syndrome. Cereb Cortex. 2016; 26(5): 2084–2092. PubMed Abstract \| Publisher Full Text\n\nVentura R, Pascucci T, Catania MV, et al.: Object recognition impairment in Fmr1 knockout mice is reversed by amphetamine: involvement of dopamine in the medial prefrontal cortex. Behav Pharmacol. 2004; 15(5–6): 433–442. PubMed Abstract \| Publisher Full Text\n\nde Bruin JP, Sànchez-Santed F, Heinsbroek RP, et al.: A behavioural analysis of rats with damage to the medial prefrontal cortex using the morris water maze: evidence for behavioural flexibility, but not for impaired spatial navigation. Brain Res. 1994; 652(2): 323–333. PubMed Abstract \| Publisher Full Text\n\nRomano E, Cosentino L, Laviola G, et al.: Genes and sex hormones interaction in neurodevelopmental disorders. Neurosci Biobehav Rev. 2016; 67: 9–24. PubMed Abstract \| Publisher Full Text\n\nReynolds CD, Nolan SO, Jefferson T, et al.: Sex-specific and genotype-specific differences in vocalization development in Fmr1 knockout mice. Neuroreport. 2016; 27(18): 1331–1335. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNolan SO, Lugo JN: Dataset 1 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source\n\nNolan SO, Lugo JN: Dataset 2 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source\n\nNolan SO, Lugo JN: Dataset 3 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source\n\nNolan SO, Lugo JN: Dataset 4 in: Reversal learning paradigm reveals deficits in cognitive flexibility in the Fmr1 knockout male mouse. F1000Research. 2018. Data Source" }	[ { "id": "34794", "date": "20 Jun 2018", "name": "Laura Smith", "expertise": [ "Reviewer Expertise fragile X", "autism", "addiction", "dendritic structure and plasticity" ], "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 authored by Nolan and Lugo investigates the ability of Fmr1 KO mice on the FVB.129 background to demonstrate proper reversal learning in the Morris water maze (MWM). Background strain appears to make a large difference in observed cognitive deficits in mice lacking expression of the Fmr1 gene, and this study makes a valuable contribution to the literature by assessing MWM reversal learning in Fmr1 KO mice on the pure FVB background.\n\nSuggested revisions:\nThe authors refer to papers showing impaired reversal learning in Fmr1 KO mice on the C57BL/6J strain and cite D’Hooge et al., 19971 and Kooy et al., 19962 However, in each of these studies the authors specify that only pigmented offspring were selected for testing, suggesting that these mouse lines were not fully backcrossed to C57BL/6J. Of note, stem cells used in the creation of transgenic mice have often been derived from the 129 strain. In any case, the authors should likely revisit this statement, and instead may wish to report Paradee et al. (1999)3 which used fully backcrossed C57BL/6 KO mice. The statement that “few studies have examined cognitive flexibility in a reversal form of the MWM task” (presumably in Fmr1 KO mice) seems inaccurate, as most studies using MWM to assess Fmr1 KO mice actually include this task (Paradee et al., 1999; Kooy et al., 1996; D’Hooge et al., 1997; The Dutch-Belgian Fragile X Consortium paper, 19944; Baker et al., 2010)5. However, as the authors state, it had not been properly assessed on the FVB background. The abstract conclusion says that “previous studies have not demonstrated deficits in spatial memory in the Fmr1 KO model.” However, multiple previous assessments of Fmr1 KO mice in the MWM have shown minor acquisition differences compared to WT mice (e.g., Paradee et al., 1999; Kooy et al., 1996, Am J Med Genet; The Dutch-Belgian Fragile X Consortium paper, 1994). Please consider these and also deficits observed in the plus-shaped water maze (e.g., Dobkin et al., 20006; Van Dam et al., 20007) and radial arm maze (Mineur et al., 20028) to perhaps soften this statement appropriately. Consider changing “disability” to “disabilities” in first sentence of the abstract. Please add more detail about the Morris water maze task. For instance, did mice receive consecutive trials within each block? Was entry quadrant varied or static over each mouse’s trials within (and across) blocks? Was the temperature of the water controlled? What were the visual cues, where were they positioned with respect to each quadrant, and were they proximal or distal? If animals were artificially colored for detection in the maze, please specify. Were animals dried and/or warmed between or after trials? It may be helpful to label days on Fig. 1B. In the first Results paragraph-- in the absence of a significant main effect or interaction involving genotype, further independent samples t-testing between WT and KO should probably not be reported. In the Discussion, the authors point to deficits in Fmr1 KO prefrontal cortex LTP with regard to the observed reversal learning deficit, which is fine. However, using this same logic to claim support for lack of observed differences in acquisition, based on findings that PFC is not relevant to acquisition, is somewhat flawed. It may be more appropriate (e.g., in the sentence regarding PFC and learning a new location) to simply acknowledge that \"PFC is not required for acquisition\" and allow the reader to intuit the point (that seems to be intended) more subtly. If this topic will be discussed, other brain regions likely involved in MWM acquisition (e.g., hippocampus), and that also show significant plasticity-related variations in Fmr1 KO compared to WT mice, should be included. The authors’ point that this investigation should be made in females is well-received. However, it is not immediately clear how these data in male Fmr1 KO mice provide “preliminary evidence that could be applied to tease apart subtle differences between the male and female Fmr1 KO phenotype.” Perhaps this point can be further clarified or removed? In the attached data sets, it is not clear why a few animal IDs are repeated. Specifically, please see the “Learning trial” set, and the “transformed” data on the right-hand side of the screen. Subjects 124 and 134 are listed twice, but all other IDs appear once. Also, a little more explanation, either directly on the data sheets or elsewhere in the manuscript, for the trial names, etc. seen here may be beneficial to users of these data.\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": [] }, { "id": "34795", "date": "02 Jul 2018", "name": "Andre Fenton", "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 am afraid that I did not find this manuscript and the results to be compelling. The experimental design is straightforward but the analysis is questionable and the report is not as scholarly as it should be.\nStatistics are used questionably. 1) t tests are used for post-hoc comparisons, when they are not justified, For example, post-hoc t tests are performed after ANOVA has failed to find relevant differences. 2) Furthermore, the alpha level for post-hoc t tests seem not to be corrected for multiple comparisons. The reported significant difference on trial 3 may not maintain after correcting alpha for multiple comparisons 0.05/4, although it might - a reader can’t know. Regardless, what is the justification for the t tests after the ANOVA results were not significant?\nDataset 1: Given no effects of genotype or interaction in the ANOVA, what justifies data snooping as follows: \"Further independent samples t-testing revealed no differences between WT and KO at any of the 8 different trials, p > 0.05.” ?\n\nDataset 2: Given the ANOVA was not significant for a 1-way ANOVA, there is no justification for a t test and in fact it is numerically equivalent to the F-test, so even more so not warranted. \"Further independent samples t-testing revealed no differences in duration in any of the quadrants, p > 0.05.”\nThe manuscript states that \"Fmr1 KO mice showed deficits in their ability to learn the new location of the platform, Fgenotype (1, 23) = 3.93, p = 0.059.” Why is p= 0.059 indicative of a deficit?\nThe Introduction is written as if the water maze is the only way to test spatial memory and cognitive flexibility, when of course it is not.\nThe Discussion states \"The current study did, however, demonstrate impairments in cognitive flexibility in the Fmr1 KO. As previously mentioned, other studies have not before detected such changes“ Remarkably, the authors fail to consider the work from the Fenton lab which explicitly has investigated cognitive flexibility in Fmr1 KO mice (Radwan et al., 2015 Neurobiol. Dis.; Dvorak et al., 2018, PLoS Biol1.) along with electrophysiological correlates of the ability.\nSomething is wrong with the initial statement: \"Male Fmr1+/+ and female Fmr1+/- FVB.129P2-Pde6b+Tyrc-ch Fmr1tm1Cgr/J (Stock No: 004624, The Jackson Laboratory, Bar Harbor, ME, USA) mice were used as breeders (9 total breeding pairs) to produce the following groups:” because WT mice cannot contribute to breeding involving a KO male.\nFig 1B should have the days indicated, as well as the rest period.\nFig. 2C shows poor learning even in WT, despite the significant effect of trial. Could the paradigm simply not be robust in mice? There is a literature on this possibility, see Wolfer et al., 1998 News Physiol Sci2.).\nWhat justification is there for the assertion that \"Moreover, this ability to adapt to a new location is mediated through multi-synaptic connections between the hippocampus and the prefrontal cortex13.”? What is the mechanism that is referenced by the statement \"This proposed mechanism further supports our findings of no change to the initial spatial learning phase, as lesions to this area did not impact initial learning performance in spatial navigation11.”?\nThe final statement seems untrue given that the authors claim to find a reversal deficit but say there had not been one detected previously \"Overall, this study corroborates and extends previous evidence of impaired cognitive flexibility in the male Fmr1 KO mouse.”\nWhat is \"t-testing\" should this not simply be stated as \"t tests” ?\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? No", "responses": [] }, { "id": "34793", "date": "16 Jul 2018", "name": "Lisa Monteggia", "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 current study examines male Fmr1 knockout mice in the Morris Water maze. This group focuses on the FVB strain as previous work has shown background strain differences can impact the phenotype of Fmr1 knockouts. The authors show that the male Fmr1 knockout mice have no impairment in acquisition of spatial memory but do show differences in the reversal learning paradigm that are not attributed to vision or motor differences. In the reversal learning paradigm, the authors show that during the 4 blocks of reversal learning the Fmr1 KOs show a rather stable latency to find the platform in contrast to the WT mice that show the expected decrease to reach the platform with increasing trials. The authors highlight the finding that male Fmr1 knockout mice have deficits in reversal learning as assessed in the Morris water maze but not in acquisition, demonstrating a link to a specific form of learning in this paradigm. The study is straightforward and the data clearly presented.\n\nA couple of comments the authors should further clarify: The authors should elaborate on the breeding scheme as they are not directly comparing littermate controls.\n\nBased on the authors discussion point regarding potential sex differences between male and female Fmr1 KO mice, is there data to suggest differences in cognitive flexibility between the males and female KOs?\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-711
https://f1000research.com/articles/7-708/v1	07 Jun 18	{ "type": "Research Article", "title": "Continuous positive airway pressure plus low flow oxygen versus usual care of severe acute cardiogenic pulmonary edema in the pre-hospital setting: A randomised controlled trial", "authors": [ "Michael A. Austin", "Karen Wills", "David Kilpatrick", "E. Haydn Walters", "Karen Wills", "David Kilpatrick", "E. Haydn Walters" ], "abstract": "Background: Acute cardiogenic pulmonary edema (ACPE) is characterized by acute breathlessness and hypoxia and is associated with poor prognosis. Standard pre-hospital management of ACPE includes high-flow oxygen, nitroglycerin and, in severe cases, assisted ventilation. Patients with ACPE can be supported with newer modalities of non-invasive ventilation, specifically continuous positive airway pressure (CPAP). The aim of this study was to determine whether patients with ACPE treated with CPAP plus low-flow oxygen pre-hospitally have a lower mortality rate than those treated conventionally. Methods: This study was a pre-hospital randomised, non-blinded controlled trial conducted July 2009–July 2010. Included were all participants transported by ambulance and admitted to the Royal Hobart Hospital, Tasmania, Australia. The study population was consecutive persons ≥18 years of age with sudden onset of severe respiratory distress, diagnosed as ACPE. Patients were included if they required ventilatory assistance. Patients required a GCS >12 and blood pressure >90 mmHg systolic to safely receive CPAP. The primary outcome was pre- or in-hospital mortality. Results: In total, 50 patients were enrolled with mean age of 79.8 (±11.9) years. There were two deaths (8.3%) in the CPAP arm and nine (34.6%) in the control arm (RR, −0.24; 95% CI, 0.06–1.00; p=0.051) with a number needed to treat of 4. CPAP plus low-flow oxygen was significantly less likely to result in respiratory acidosis (mean difference in pH, −0.11; 95% CI, −0.04–−0.17; p=0.002), with elevated pCO2 (mean difference, −10.0 mmHg; 95% CI, −19.2–−0.78; p=0.026). The length of hospital stay was significantly shorter in the surviving patients who received CPAP (ratio of means, 0.45; 95% CI, 0.29–0.70; p≤0.001). Discussion: This study, which provides interim results due to early termination of the trial, shows CPAP in the pre-hospital setting for ACPE is practicable and is associated with improved patient outcomes.", "keywords": [ "CPAP", "pulmonary edema", "ambulance", "NIPPV", "out-of-hospital", "pre-hospital" ], "content": "Introduction\n\nCongestive heart failure occurred in 5.7 million Americans1, and in 10 million Europeans2. In the United States, 670,000 new cases of acute exacerbations of heart failure (AHF) are diagnosed each year1. Unlike other cardiovascular conditions, it is increasing in incidence and prevalence3. Its management imposes a substantial burden on the health care system, accounting for 1–2% of total healthcare costs in industrialized nations4. About 70% of these costs are related to hospitalization4. Acute cardiogenic pulmonary oedema (ACPE) is characterized by acute breathlessness and hypoxia5, and is associated with poor prognosis5. Standard pre-hospital management of ACPE in most ambulance/paramedic services throughout the world includes high-flow oxygen, nitroglycerin and, in severe cases, assisted ventilation with a bag valve mask or endotracheal intubation (ETI)6. Some services use frusemide as a diuretic and morphine at low dose as an anxiolytic but these are controversial6.\n\nInvasive ventilation increases the risk of complications, including hospital acquired infection (HAI) (pneumonia, sinusitis) and tracheal injury, and consequently may prolong ICU and hospital stay6–8. Pre-hospital intubation is especially difficult in this patient population and is not routinely part of pre-hospital protocols. Therefore in the subset of patients with severe ACPE who are not responding to oxygen and medical therapy, ventilatory assistance is first given non-invasively, conventionally with a valved bag and mask (bagging)6. However, bagging with a bag and mask in the pre-hospital setting can also have inherent risks: it is difficult to synchronize with the patient’s respiratory efforts while moving in an ambulance, and trying to provide adequate assisted ventilation this way may be counter-productive9. Patients with ACPE can be supported with newer modalities of non-invasive positive pressure ventilation (NPPV), specifically continuous positive airway pressure (CPAP). In itself this improves ventilation and therefore oxygenation, through improved V/Q mismatch, reduced breathing effort and increased cardiac output by reducing left ventricular after-load10–13.\n\nDespite the potential advantages of CPAP for the management of severe ACPE14, there is a lack of high-quality trial evidence evaluating its impact on mortality in its use in the pre-hospital setting. The hypothesis of this randomised clinical trial was that the use of CPAP system would reduce mortality, hospital length of stay and the requirement for intubation for such patients compared with conventional care provided in the pre-hospital setting.\n\n\nMethods\n\nThis study has been reported according to the CONSORT guidelines. A completed checklist can be found in Supplementary File 1.\n\nThe full trial protocol can be found in Supplementary File 2.\n\nThis randomised, controlled, parallel-group trial, which was non-blinded but with good concealment of allocation, was undertaken in patients with a diagnosis of severe ACPE from July 2009 to July 2010. The Joint Tasmanian Ethics Committee (Tasmania Health & Medical Human Research Ethics Committee; EC00337) approved the study with patient consent waived (Ethics Reference Number: H0010364), as in such an emergent therapeutic area both treatments were accepted practices, time was of the essence and neither the patients nor relatives were in a position to make informed choices. The study was registered with the Australia New Zealand Clinical Trials Registry (Trial ID, ACTRN12609000410257).\n\nThe RCT consisted of two treatment arms: a control arm using standard positive pressure ventilation with high flow oxygen with a bag-valve-mask assisting patients’ ventilations (bagging), versus an active arm using a continuous positive airway pressure (CPAP) device (Whisperflow) and oxygen supplied at 28–33%. All participants were transported by ambulance to the Emergency Department of the Royal Hobart Hospital (RHH). The RHH is the tertiary hospital for the state of Tasmania and is the only major acute public general hospital serving a population of 250,000 in Southern Tasmania, Australia. No changes to the trial were made after commencement.\n\nPatients were included if they were ≥18 years of age and diagnosed by paramedic ambulance staff with severe ACPE requiring ventilator assistance. Patients were excluded if they were in respiratory or cardiac arrest, had a Glasgow Coma Score (GCS) <12 or had a systolic blood pressure <90 mmHg.\n\nInitially, all subjects received standard therapy according to Ambulance Tasmania (AT) guidelines, namely initial high-flow oxygen, sublingual nitroglycerin as a primary therapy in increasing incremental doses from 400–1600 µg, increasing every 5 min as long as systolic blood pressure was >100 mmHg, with 1–2 mg IV morphine as an anxiolytic and 40 mg IV frusemide for severe respiratory distress in patients with a transport time greater than 20 min.\n\nWe used computerised random number generation to allocate patients to treatment arms. This procedure ensured that treatment allocation was concealed before randomisation. Neither paramedics nor the research team were blinded to treatment after randomisation. Subjects entered the study and were randomised at the point where the ambulance personnel considered that they required non-invasive ventilatory assistance. This could be immediately on initial contact or after some standard therapy but subsequent deterioration. Patients in the active arm received 10 cm of H2O CPAP delivered by Whisperflow® (flow created by a mix of oxygen and air delivering a fixed oxygen flow rate providing 28–33% oxygen), with continued concurrent therapy in the AT Protocol for ACPE patients. In the control arm, positive pressure ventilation was provided by a conventional bag-valve-mask system with high flow oxygen at a rate of 8–15 l/min (bagging), again along with the same concurrent per-protocol therapy. Pulse oximeters were used to measure oxygen saturations in both groups. Baseline and follow-up measurements of oxygen saturation and vital signs, including GCS scores were recorded on the computerized Ambulance Report Form (Victorian Ambulance Clinical Information System). Vital signs recorded at the time of initial assessment were used as baseline measures.\n\nIn total, 98% (62/63) of paramedics from AT agreed to participate and were trained in the use of the CPAP device and the study protocol. Randomization and allocation concealment were achieved using computer-generated numbers printed on cards indicating the assigned device, which were sealed in sequentially numbered opaque envelopes placed with the ventilation/CPAP equipment on every ambulance.\n\nHaving identified a suitable study patient, the paramedic opened the next envelope and entered the randomization number into the computerized ambulance reporting system. A list of these patients was sent to the study coordinator each week. As is standard with such severe patients, they were pre-notified to the Emergency Department (ED) at RHH as an impending Category 1 (high priority) patient, but also that the patient should be recorded as part of the CPAP trial. At arrival at the ED, paramedics were asked to request ED personnel to follow the study protocol and to draw arterial blood gas (ABG) within 30 min; the study was then complete and conventional patient care continued under the treating emergency physician.\n\nThe randomisation cards were collected and accounted for by the research team (MA and MG) shortly after each use. The team also followed up each set of ambulance case notes to ensure that the correct device had indeed been used, since neither paramedics nor the research team were blinded to treatment after allocation. In addition, a full list of patients during the study period who had been transported by ambulance with an ED and hospital discharge diagnosis of ACPE was collected, and case notes were assessed to determine if any patients who either required ventilatory assistance or who received it were missed during this time.\n\nTwo independent physicians blinded to group allocation retrospectively reviewed randomised study patient computerised ambulance and hospital records to confirm that they had met the set diagnostic criteria for severe ACPE (crackles on auscultation, acute onset shortness of breath and hypoxia requiring assisted ventilation); where appropriate, the cause of mortality was also confirmed from the notes and/or death certification. Discrepancies were reviewed by a third physician (EHW) who was blinded to treatment allocation for resolution. Hospital admission data, including arterial blood gas results were obtained from the RHH Patient Information Medical System. Dispatch, arrival and transport data were obtained from dispatch records from AT to calculate length of pre-hospital treatment times. The third physician (EHW) also reviewed the hospital clinical records of those patients who died, while blinded to the treatment arm, to assess at what point critical deteriorations had occurred and crucial management decisions had been made. AT provided in-kind support for consumables, paramedic training and IT, but unfortunately pulled out at the 12-month time point for budgetary reasons, owing to the expense of CPAP consumables.\n\nThe primary outcome measure was pre-hospital or in-hospital mortality. The secondary outcomes were: requirement for invasive ventilation, arterial blood gas values upon arrival in the ED (pH, PO2, PCO2, bicarbonate), length of hospital stay (days) for patients who survived their admission to hospital, and vital signs (oxygen saturation, heart rate, blood pressure, respiratory rate and GCS score).\n\nWhere only venous blood samples were available for blood gas assessments we used published formulations to estimate corresponding arterial values for pH, carbon dioxide, and bicarbonate15. Arterial and converted venous blood gases were compared using Student’s t-tests, and if no difference was detected they were combined for subsequent analyses. It was not possible to convert venous paO2 since respective arterial paO2 values are not directly comparable and so not provided in the conversion equations, so only measured arterial PaO2 values were analysed.\n\nPower calculations for the primary outcome measure were based on the findings of Hubble’s work16, who report a mortality rate, although in patients not as severely ill as ours, of 5.4% for ACPE patients receiving CPAP compared with 23.2% for patients receiving standard therapy. A total sample size of 74 participants (37 in each group) would thus provide 80% power to detect this 17.8% absolute reduction in mortality in the CPAP arm. It was estimated from a retrospective review that at least 50 patients per year with a diagnosis of ACPE requiring ventilatory assistance were transported by AT to the RHH; therefore, the trial was intended to run for 18 months (see below). Unlike more conventional randomized controlled trials, it was not envisaged that there would be drop-outs after recruitment, between randomization and arrival, dead or alive, at the ED.\n\nBaseline characteristics for the patient cohort were described using frequencies for categorical variables and mean (±SD) for continuous variables. We used log binomial regression to compare the risk of death for patients in the two treatment arms. For the secondary outcomes, linear regression was used to compare post-treatment vital signs after adjustment for pre-treatment measurement, and to compare blood gas measurements. Non-normally distributed data were transformed as required. Variables that could not adequately be transformed were analysed using the Mann–Whitney test. Negative binomial regression was used to compare length of hospital stay for patients who survived, with the effect estimate presented as a ratio of mean length of stay for the intervention arm relative to the control arm. A ratio less than one would indicate a decrease in length of hospital stay associated with the intervention, whilst a ratio greater than one would indicate an increase. We summarized pre-hospital drug treatments as frequencies and percentages of patients treated with nitroglycerin, morphine or frusemide, and the number of doses received. Only intention-to-treat analyses were performed. Stata/IC version 12.117 was used for all analyses and a P-value of 0.05 was considered statistically significant.\n\n\nResults\n\nA total of 377 patients with varying severity of ACPE were treated and transported to the RHH during the first 12 months, before the study was prematurely terminated as a result of budgetary concerns from AT. Of these, 50 (13%) met the inclusion criteria and were considered eligible for inclusion in the study according to the attending paramedics: 26 were assigned to the conventional bagging arm, and 24 to the CPAP arm (Figure 1). Of the remaining 327 patients transported, none received CPAP or bagging, so no missing patients were identified (Figure 1). All patients received treatment with the allocated device. Although the intended recruitment numbers were not achieved, the CIs decided to continue with the analysis as planned to avoid publication bias and to ensure that the data are available for meta-analysis. Thus, the analysis was completed on an intention-to-treat basis with the numbers obtained. Baseline characteristics in the two groups were similar for age, initial oxygen saturation, respiratory rate, blood pressure, heart rate, GCS score and pre-hospital treatment time; however, there were more females in the CPAP arm (Table 1). ED diagnosis confirmed the opinion of the ambulance personnel for all patients enrolled as ACPE, and this was also endorsed by a later review of investigator notes. The median treatment time (scene arrival to ED) was 35 mins.\n\nUnless otherwise indicated.\n\nWe found modest evidence for a reduction in overall mortality in the treatment arm (RR, 0.24; 95% CI, 0.06–1.00; p=0.051; Table 2) from 34.6% (9 deaths) in the bagging arm to 8.3% (2 deaths) in the CPAP arm. This represents a 76% relative reduction in mortality. There were 11 deaths, all in hospital, with 8 occurring in the first 24 hours. Of these deaths, nine were due to a cardiac cause (essentially heart failure), one patient died of a stroke, and the final death at 9 days was due to an unexpected abdominal catastrophe (volvulus and secondary necrosis).\n\nIntention-to-treat analysis.\n\nUnless otherwise indicated; †relative risk; §incidence rate ratio; #chi-squared statistic; ‡mean difference.\n\nThe mean (SD) length of hospital stay was 7.2 (5.11) days for surviving patients in the control arm (n=17) and 3.23 (2.37) days for patients in the CPAP arm (n=22). From the regression analysis (Table 2), it is estimated that the expected length of hospital stay would decrease by 55% with CPAP compared with usual care (ratio of means, 0.45; 95% CI, 0.29–0.70; p≤0.001). Blood gas analysis was undertaken within 30 minutes in 78% (39/50) patients; 48% (24/50) were arterial and 30% (15/50) venous. Patients receiving CPAP were significantly less likely to have acidosis (pH <7.35) and hypercarbia (PaCO2 >45 mmHg) (Table 2); the hospital arrival post-ambulance-treatment oxygen saturation of patients was significantly lower in those who received CPAP (Table 3). Only two patients were intubated in the ED, both having been bagged, and both died in the ED. There were no significant differences in ED arrival heart rate, blood pressure and GCS between the two study arms (Table 3).\n\n*Unless otherwise indicated; †regression coefficient adjusted for pre-treatment measurement; #Mann–Whitney test z-score.\n\nUse of out-of-hospital medications was similar between the two groups: sublingual nitroglycerin was used in 73% (19/26) of patients in the control arm and all patients (24/24) in the CPAP arm; frusemide was used in 50% (13/26) of patients in the control arm, and in 75% (18/24) of patients in the CPAP arm; low-dose morphine was used in 23% (6/26) of patients in the control arm and 25% (6/24) of patients in the CPAP arm. Of those patients receiving drug treatment, the median (IQR) total doses for sublingual nitroglycerin sprays (400 ug/spray) was 2.8 sprays (2.8) for the control arm and 2 sprays (2.8) for the CPAP arm. For frusemide, most patients (92% in the control arm and 89% in the CPAP arm) received a total dosage of 40 mg, one in each arm received 20 mg and one in the CPAP arm received 80 mg. Dosage data were recorded for 9/12 patients receiving morphine: the range of total dosage in the control arm was 1–3 mg and was 1–2 mg in the CPAP arm.\n\n\nDiscussion\n\nWe found that treatment with CPAP plus low-flow oxygen, in the pre-hospital setting, for patients with ACPE reduced the risk of death by 76% compared with conventional care. Of the 377 patients transported to hospital with ACPE during the year of the study, we recruited all those eligible in the most severe group requiring assisted ventilation. The difference in mortality between the active and control groups was greater than anticipated, because of a larger than expected number of deaths in the control group. This may be due to the greater severity of patient condition at the time of randomisation in our study compared to that used as the basis for the power calculation16. Although baseline vital signs were not different between groups, patients were more likely to have respiratory acidosis with conventional care, and it may be construed that conventional care may have contributed to this increase in mortality.\n\nAlthough the overall number recruited was smaller than intended, for the logistic ambulance service managerial decision given, our results, are consistent with most of the limited pre-hospital literature11,12,16,18,19. Two recent before-and-after studies showed no differences in mortality, although the population studied had less severe acute exacerbations of heart failure20,21. The population around Hobart is reasonably urbanized, but there are significant outlying rural areas, offering a mixed population, which makes our study reasonably generalizable for other similar urban rural setting. Whilst we found evidence for a difference in mortality between the management strategies trialled, we did not recruit the required sample size so did not reach the required level of power. This investigation should perhaps be best regarded as preliminary and of a “pilot” nature. Although, by chance, we had an imbalance in the sex composition of the treatment groups, there is no evidence to indicate an association between sex and mortality, so it is unlikely to have been a confounding factor.\n\nA second limitation of this study was that decision-making about patient eligibility was left to the discretion of the paramedics on the scene, with no validated physiological or clinical scores used22,23. However, given that the paramedic did not know which regimen would be allocated at the time of decision to enrol each patient, this approach is unlikely to have confounded the outcome. Studies have shown that field discrimination between ACPE and other causes of respiratory distress is poor16, even by in-field physicians24. However, our review of hospital records for all patients with heart failure who were transported to hospital over this period indicated that all suitable patients were randomised, and all randomised patients had an admission diagnosis of ACPE and received the allocated treatment. It does not appear that any suitable patients were missing from the recruitment.\n\nThe rate of arterial blood gas sampling for study patients was low, with only 48% of arterial samples drawn within 30 minutes of arrival. We recognize that ABGs are not standard of care; however, with the variability of VBG results, we felt ABGs would be a better lab value of respiratory status. Despite the request by research staff in meetings leading up to the study, this rate of compliance by hospital staff is consistent with the literature25,26. Potential reasons for this low compliance include patient refusal, reluctance of doctors to perform the test or be involved in other investigators’ research and limited time and staff resources20,25. Conversion of VBG samples is also a limitation of our study. Arterial and converted venous blood gases were compared using Student’s t-tests and no significant group differences were found. In addition, a sensitivity analysis using only the ABG results showed similar results. Therefore, we feel confident in combining these data.\n\nWe were unable to determine whether pre-hospital management itself had an effect on in-hospital management, except perhaps that related to the presenting state of the patient. The RHH has a BiPAP/CPAP protocol for breathless patients who present with severe respiratory distress with a clinical picture of ACPE, but any change in management that occurred after arrival at the ED would have reduced the differences between the treatment arms. However, the hospital chart review suggested that those who died tended to do so quite quickly after arrival in hospital.\n\nThe number needed to treat with CPAP to avoid a cardiac-related death was 4. Our mortality rate in the CPAP arm was consistent with published data11,12,16. Notably, our study provides additional evidence to underpin recent recommendations that CPAP be used in the pre-hospital setting for severe ACPE patients11,12,27. However, the use of controlled oxygen delivered with CPAP in this study is a potential confounder. Thus, we cannot be sure whether the advantage in the active group was through the direct mechanical effects of CPAP, different oxygen regimes in the two systems used, or both. Previous results with CPAP in patients with milder heart failure would certainly support the use of this modality28. It would probably not be ethical to undertake a trail of CPAP versus low-flow oxygen at this stage.\n\nOverall, carbon dioxide blood levels were higher in the “bagging” group, with secondary acidosis, indicating relatively worse ventilation, while the oxygen saturations were higher; this raised the question of whether relative hyperoxia was contributing to a V/Q mismatch or suppressing ventilation in a vulnerable group, or whether both are merely features of the respective systems being used. The relative hypercarbia and acidosis when patients were bagged with high-flow oxygen (Table 3) is consistent with that seen in previous hospital studies29. Supporting the use of CPAP in these patients.\n\nThe overall incidence of endotracheal intubation was low, and was nil in patients with CPAP use, which is consistent with the findings of Thompson et al.11. However, this raises the question of why those that died were not intubated, especially on arrival in extremis in the ED. Review of the ED notes suggested that the commonest scenario in those who died was an elderly patient presenting with severe heart failure and ventilatory failure being trialled on BiPAP in the ED, with concurrent discussions taking place with family about the decision not to progress to intubation and mechanical ventilation. With family agreement, the patient was then allowed to die comfortably (i.e. a palliative approach was taken). None of the patients had documented “do not resuscitate” advanced directives prior to arrival to the ED. From the baseline characteristics, the severity of the ACPE event appeared similar in each group, so use of the CPAP regime appeared to have prevented this scenario. This strongly indicates that optimal pre-hospital treatment is vital to prevent clinical deterioration to a point where the ED staff are faced with what is essentially an “end-stage” patient thought unsuitable for intubation and ICU care. The length of hospital stay in survivors was significantly longer in the control group compared to the CPAP group, consistent with both the literature12 and with CPAP allowing patients to arrive in hospital in a better physiological state.\n\n\nConclusion\n\nIn conclusion, our trial, although truncated, found that pre-hospital CPAP plus low-flow oxygen resulted in a 76% relative reduction in the risk of mortality for severe ACPE patients compared with the conventional form of ambulance service management with assisted mask and bag ventilation. Patients that underwent CPAP were less likely to have respiratory acidosis and their length of hospital stay was significantly reduced. Our findings provide evidence to support CPAP with controlled oxygen in severe ACPE patients in the pre-hospital setting11,12. Given the early termination of this study, a further trial of this combination should be considered reasonable.\n\n\nData availability\n\nDataset 1. All raw demographic and experimental data from the present study. DOI: 10.5256/f1000research.14577.d20129230.", "appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe authors declare that no grants were involved in supporting this work. Fisher and Paykal (suppliers of the Whisperflow® CPAP device) had no involvement in the study design, conduct, analysis or interpretation of data, nor in writing this report. The NHMRC through the CRE supported the statistician and co-investigator KW. Ambulance Tasmania provided in-kind support for consumables, paramedic training and IT, but for budgetary reasons, due to the expense of CPAP consumables, pulled out at the 12-month time point. AT did not have access to the study data and had received no information about study progress when they withdrew support. Furthermore, the trial investigators had no involvement in or prior knowledge of the decision.\n\n\nAcknowledgements\n\nWe would like to thank the patients who participated in the study, clinical and clerical staff of Ambulance Tasmania and the Emergency Department at the Royal Hobart Hospital, laboratory staff at the Royal Hobart Hospital and members of the Respiratory Department at the Royal Hobart Hospital whose participation made this project possible.\n\n\nSupplementary materials\n\nSupplementary File 1. Completed CONSORT checklist.\n\nClick here to access the data.\n\nSupplementary File 2. Research protocol for the trial.\n\nClick here to access the data.\n\n\nReferences\n\nRoger VL, Go AS, Lloyd-Jones DM, et al.: Heart disease and stroke statistics--2011 update: a report from the American Heart Association. Circulation. 2011; 123(4): e18–e209. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNieminen MS, Böhm M, Cowie MR, et al.: Executive summary of the guidelines on the diagnosis and treatment of acute heart failure: the Task Force on Acute Heart Failure of the European Society of Cardiology. Eur Heart J. 2005; 26(4): 384–416. PubMed Abstract \| Publisher Full Text\n\nSchlosshan D, Elliott M: Prognostic indicators in patients presenting with acute cardiogenic pulmonary edema treated with CPAP: it's not the acid that matters, it's back to basics. Crit Care. 2010; 14(6): 1009. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStewart S, Blue L, Walker A, et al.: An economic analysis of specialist heart failure nurse management in the UK; can we afford not to implement it? Eur Heart J. 2002; 23(17): 1369–78. PubMed Abstract \| Publisher Full Text\n\nFranciosa JA, Wilen M, Ziesche S, et al.: Survival in men with severe chronic left ventricular failure due to either coronary heart disease or idiopathic dilated cardiomyopathy. Am J Cardiol. 1983; 51(5): 831–6. PubMed Abstract \| Publisher Full Text\n\nPetrie DA, CE, Tallon JM, et al.: Evidence based protocols. Emergency Health Services Nova Scotia. 2013. Reference Source\n\nGirou E, Brun-Buisson C, Taillé S, et al.: Secular trends in nosocomial infections and mortality associated with noninvasive ventilation in patients with exacerbation of COPD and pulmonary edema. JAMA. 2003; 290(22): 2985–91. PubMed Abstract \| Publisher Full Text\n\nLevitt MA: A prospective, randomized trial of BiPAP in severe acute congestive heart failure. J Emerg Med. 2001; 21(4): 363–9. PubMed Abstract \| Publisher Full Text\n\nMehta S, Jay GD, Woolard RH, et al.: Randomized, prospective trial of bilevel versus continuous positive airway pressure in acute pulmonary edema. Crit Care Med. 1997; 25(4): 620–8. PubMed Abstract \| Publisher Full Text\n\nWilliams JF, Bristow MR, Fowler MB, et al.: Guidelines for the evaluation and management of heart failure. Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee on Evaluation and Management of Heart Failure). J Am Coll Cardiol. 1995; 26(5): 1376–98. PubMed Abstract \| Publisher Full Text\n\nThompson J, Petrie DA, Ackroyd-Stolarz S, et al.: Out-of-hospital continuous positive airway pressure ventilation versus usual care in acute respiratory failure: a randomized controlled trial. Ann Emerg Med. 2008; 52(3): 232–41, 241.e1. PubMed Abstract \| Publisher Full Text\n\nVital FM, Saconato H, Ladeira MT, et al.: Non-invasive positive pressure ventilation (CPAP or bilevel NPPV) for cardiogenic pulmonary edema. Cochrane Database Syst Rev. 2008; (3): CD005351. PubMed Abstract \| Publisher Full Text\n\nKeenan SP, Kernerman PD, Cook DJ, et al.: Effect of noninvasive positive pressure ventilation on mortality in patients admitted with acute respiratory failure: A meta-analysis. Crit Care Med. 1997; 25(10): 1685–92. PubMed Abstract \| Publisher Full Text\n\nDucros L, Logeart D, Vicaut E, et al.: CPAP for acute cardiogenic pulmonary oedema from out-of-hospital to cardiac intensive care unit: a randomised multicentre study. Intensive Care Med. 2011; 37(9): 1501–9. PubMed Abstract \| Publisher Full Text\n\nAk A, Ogun CO, Bayir A, et al.: Prediction of arterial blood gas values from venous blood gas values in patients with acute exacerbation of chronic obstructive pulmonary disease. Tohoku J Exp Med. 2006; 210(4): 285–90. PubMed Abstract \| Publisher Full Text\n\nHubble MW, Richards ME, Jarvis R, et al.: Effectiveness of prehospital continuous positive airway pressure in the management of acute pulmonary edema. Prehosp Emerg Care. 2006; 10(4): 430–9. PubMed Abstract \| Publisher Full Text\n\nStataCorp: Stata Statistical Software: Release 10. College Station, TX. 2007.\n\nSen A: ACP Journal Club: review: in acute respiratory failure, prehospital CPAP reduces mortality and intubation rates. Ann Intern Med. 2015; 162(8): JC5. PubMed Abstract \| Publisher Full Text\n\nKnox N, Chinwe O, Themba N, et al.: Relationship between intubation rate and continuous positive airway pressure therapy in the prehospital setting. World J Emerg Med. 2015; 6(1): 60–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrew Willmore RD, Maloney J, Ouston E, et al.: Effectiveness and safety of a prehospital program of continuous positive airway pressure (CPAP) in an urban setting - ERRATUM. CJEM. 2015; 1 FirstView: 1-M3. PubMed Abstract \| Publisher Full Text\n\nCheskes S, Turner L, Thomson S, et al.: The Impact of Prehospital Continuous Positive Airway Pressure on the Rate of Intubation and Mortality from Acute Out-of-hospital Respiratory Emergencies. Prehosp Emerg Care. 2013; 17(4): 435–41. PubMed Abstract \| Publisher Full Text\n\nKeim SM, Spaite DW, Maio RF, et al.: Risk adjustment and outcome measures for out-of-hospital respiratory distress. Acad Emerg Med. 2004; 11(10): 1074–81. PubMed Abstract\n\nWelsford M, Morrison LJ: Defining the outcome measures for out-of-hospital trials in acute pulmonary edema. Acad Emerg Med. 2002; 9(10): 983–8. PubMed Abstract\n\nKallio T, Kuisma M, Alaspää A, et al.: The use of prehospital continuous positive airway pressure treatment in presumed acute severe pulmonary edema. Prehosp Emerg Care. 2003; 7(2): 209–13. PubMed Abstract\n\nWijesinghe M, Perrin K, Healy B, et al.: Pre-hospital oxygen therapy in acute exacerbations of chronic obstructive pulmonary disease. Intern Med J. 2011; 41(8): 618–22. PubMed Abstract \| Publisher Full Text\n\nWijesinghe M, Perrin K, Ranchord A, et al.: Routine use of oxygen in the treatment of myocardial infarction: systematic review. Heart. 2009; 95(3): 198–202. PubMed Abstract \| Publisher Full Text\n\nBritish Thoracic Society Standards of Care Committee: Non-invasive ventilation in acute respiratory failure. Thorax. 2002; 57(3): 192–211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBledsoe BE, Anderson E, Hodnick R, et al.: Low-fractional oxygen concentration continuous positive airway pressure is effective in the prehospital setting. Prehosp Emerg Care. 2012; 16(2): 217–21. PubMed Abstract \| Publisher Full Text\n\nGray AJ, Goodacre S, Newby DE, et al.: A multicentre randomised controlled trial of the use of continuous positive airway pressure and non-invasive positive pressure ventilation in the early treatment of patients presenting to the emergency department with severe acute cardiogenic pulmonary oedema: the 3CPO trial. Health Technol Assess. 2009; 13(33): 1–106. PubMed Abstract \| Publisher Full Text\n\nAustin MA, Wills K, Gibson M, et al.: Dataset 1 in: Continuous positive airway pressure plus low flow oxygen versus usual care of severe acute cardiogenic pulmonary edema in the pre-hospital setting: A randomised controlled trial. F1000Research. 2018. Data Source" }	[ { "id": "35741", "date": "28 Aug 2018", "name": "Arantxa Mas Serra", "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 was designed to investigate the impact of prehospital CPAP in the mortality of patients with cardiac acute pulmonary edema, in comparison to bag-valve-mask (BVM). Patients were elected by paramedics according to their perception of severity after protocolized medical treatment, and randomized to CPAP or BVM. Authors conclude that CPAP is better than BVM in improving respiratory acidosis and reducing mortality. Design and implementation of studies that involve interventions in prehospital setting entails a great effort of coordination, an important leadership and are difficult to carry out. My congratulations to the authors for being able to do it. In the other hand, demonstrate the impact of a short time of treatment on the overall evolution of patients is complicated and it requires studies with a large number of patients and many controlled variables.\n\nThe objective proposed in this study has been subject to some meta-analyses that have not been cited in this article. These meta-analyses concluded that the use of prehospital CPAP in patients with suspected acute pulmonary edema leads to a reduction in intubation rate and mortality of these patients. The presented results can help to do the evidence more solid.\n\nMajor considerations\n\n1. It would be interesting to discuss about the most important methodological differences with other prehospital protocols, especially regarding the respiratory support received by the control group, which does not include the possibility of intubation. As authors comment in the discussion, respiratory support with bag-valve-mask during out-of-hospital assistance in control group is not easy and could partially explain the gasometrical deterioration of these patients. BVM is not the most commonly used ventilatory support in other countries1 and its potential influence on the high mortality of the control group could be discussed. Considering that the included patients were aware (taking into account the initial vital signs), the ventilation technique could appear unnecessary. It would be interesting that authors justify in the discussion section their use instead of oxygen therapy alone in the control group. It is not clear what the clinical situation before randomization was. To expand the table 1 with vital signs and oxygen saturation at this moment would provide valuable information.\n\n2. The absence of physiological criteria of inclusion is an important limitation already commented by the authors. This issue make the study difficult to reproduce in other areas\n\n3. The authors should clarify when the data in the Dataset 1 has been collected. When studied in detail, it seems that most of the deceased patients in the control group (and one of the CPAP group) had exclusion criteria (if data marked with the number 1 are initial) such as Glasgow less than 12 or systolic blood pressure lower than 90 mmHg or unregistered. Is this interpretation correct? What does each column correspond to?\n4. I have understood that out-of-hospital treatment protocol was maintained during the first 30 minutes of hospital assistance. This issue may have caused or magnified the observed differences in the prognosis and deserves consideration in the discussion, which also includes ethical aspects of this decision. It would be interesting to report the total treatment times in both groups as well as when non invasive ventilation or CPAP was initiated in the control group, if needed. Regarding the differences in blood gases, the persistence of important respiratory acidosis in patients in the control group is not coherent with clinical situation, since vital signs at hospital arrival were not worse in the control group. It is also important to clarify whether these patients received no respiratory support on arrival and during the first 30 minutes irrespective of what their situation was (as I have understood in the methods/measurements and discussion sections), which would mean an important ethical conflict.\n\nOther considerations\n\n5. The fact that only two of the nine patients who died in the control group were finally intubate does not allow concluding about mortality, as they not received all the alternative treatment. It would be desirable to add a reference to this type of patients in the final conclusion of the article.\n\n6. Further details on the baseline cardiopathy status, severity indices and treatments received would help to conclude that the difference in mortality can be attributed to the different prehospital management. As the authors mentioned, the impact of 30 minutes in the stay of 3 to 7 days is very difficult to demonstrate and all the previous factors and significant treatments received would have to be taken into account.\n\n7. Nothing has been said about the tolerance of both methods.\n\n8. The last section of the abstract does not refer to the discussion but to the conclusions.\n9. Taking into account the method used and its limitations, the first sentence of the discussion and the conclusion could be rewritten to better reflect the results.\n\n10. The refereed study by Cheskes2 includes not only patients with less severe acute exacerbations of heart failure but also respiratory patients (almost 50% and 50%).\n\n11. Frusemide usually appears in the literature as furosemide.\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?\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? Partly", "responses": [] }, { "id": "45019", "date": "28 Mar 2019", "name": "Alix J.E. Carter", "expertise": [ "Reviewer Expertise EMS/paramedicine", "health services research" ], "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 designed randomized controlled trial about an important patient-oriented outcome (mortality). This trial had the potential to add to the literature with high quality evidence in the prehospital setting about the management of ACPE with CPAP, and it is unfortunate that the target sample size was not met due to early termination. Though the study is underpowered, the authors appropriately note that the results are promising and suggest it supports another attempt at a similar study. The results are also available for inclusion in future meta-analysis. One concern I have regarding feasibility is that the reason for early termination was the cost of the consumables for the intervention itself.\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/7-708
https://f1000research.com/articles/6-1600/v1	30 Aug 17	{ "type": "Research Article", "title": "Hepatic deletion of p110α and p85α results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity", "authors": [ "Aditi Chaudhari", "Katarina Ejeskär", "Yvonne Wettergren", "C. Ronald Kahn", "Victoria Rotter Sopasakis", "Aditi Chaudhari", "Katarina Ejeskär", "Yvonne Wettergren", "C. Ronald Kahn" ], "abstract": "Background: Class IA phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is an integral mediator of insulin signaling. The p110 catalytic and p85 regulatory subunits of PI3K are the products of separate genes, and while they come together to make the active heterodimer, they have opposing roles in insulin signaling and action. Deletion of hepatic p110α results in an impaired insulin signal and severe insulin resistance, whereas deletion of hepatic p85α results in improved insulin sensitivity due to sustained levels of phosphatidylinositol (3,4,5)-trisphosphate. Here, we created mice with combined hepatic deletion of p110α and p85α (L-DKO) to study the impact on insulin signaling and whole body glucose homeostasis. Methods: Six-week old male flox control and L-DKO mice were studied over a period of 18 weeks, during which weight and glucose levels were monitored, and glucose tolerance tests, insulin tolerance test and pyruvate tolerance test were performed. Fasting insulin, insulin signaling mediators, PI3K activity and insulin receptor substrate (IRS)1-associated phosphatidylinositol kinase activity were examined at 10 weeks. Liver, muscle and white adipose tissue weight was recorded at 10 weeks and 25 weeks. Results: The L-DKO mice showed a blunted insulin signal downstream of PI3K, developed markedly impaired glucose tolerance, hyperinsulinemia and had decreased liver and adipose tissue weights. Surprisingly, however, these mice displayed normal hepatic glucose production, normal insulin tolerance, and intact IRS1-associated phosphatidylinositol kinase activity without compensatory upregulated signaling of other classes of PI3K. Conclusions: The data demonstrate an unexpectedly overall mild metabolic phenotype of the L-DKO mice, suggesting that lipid kinases other than PI3Ks might partially compensate for the loss of p110α/p85α by signaling through other nodes than Akt/Protein Kinase B.", "keywords": [ "phosphatidylinositol-4", "5-bisphosphate 3-kinase", "p110", "p85", "insulin receptor substrate", "insulin resistance", "glucose intolerance" ], "content": "Introduction\n\nClass IA phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is a central mediator of a number of membrane receptor signaling pathways, including the insulin signaling pathway1,2. Following receptor activation by insulin, PI3K binds to tyrosine-phosphorylated amino acids of the insulin receptor substrates (IRS), resulting in PI3K activation and the formation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 has high affinity for the pleckstrin homology (PH) domain of the downstream target Akt/Protein Kinase B (PKB). The interaction of PIP3 with the PH domain enables phosphorylation of Akt/PKB by phosphoinositide dependent kinase (PDK) 1 and PDK 2, ultimately triggering a number of metabolic actions, such as lipogenesis, glycogen synthesis, inhibition of hepatic glucose output and increased glucose uptake in muscle and adipose tissue.\n\nClass IA PI3Ks consist of two subunits. The catalytic subunit, p110, contains the kinase domain responsible for the formation of PIP3. The regulatory subunit, the most common of which is p85α, binds to phosphorylated tyrosine residues in tyrosine kinases and their substrate proteins via its SH2 domain, leading to activation of PI3K activity. Both the regulatory and catalytic subunits exist as several different isoforms. In humans, there are four known catalytic subunit isoforms: p110α, p110β, p110δ and p37δ. p110α, p110β and p110δ are encoded by three different genes, PIK3CA, PIK3CB and PIK3CD, respectively (reviewed in 1), whereas p37δ (PIK3CD_v2) is a splice variant of p110δ3,4. We and others have shown that of these catalytic subunits, p110α is the major contributor for transmitting the insulin signal5–7, whereas p110β becomes active primarily in response to G protein-coupled receptor signaling and plays a role in proliferation8,9. p110δ is more cell specific than p110α and p110β, and plays an important role in immune cells and the embryonic nervous system10–12.\n\nThe regulatory subunits are also encoded by three genes, PIK3R1, PIK3R2 and PIK3R3. Their primary gene products are p85α, p85β and p55γ, respectively (reviewed in 1). PIK3R1 also encodes two splice variants of p85α, p55α and p50α, which have more limited tissue distribution. p85α is the major regulatory subunit isoform, constituting 65%–75% of the intracellular pool of regulatory subunits in most cells13. Despite the crucial role of the regulatory subunits of Class IA PI3K in mediating insulin-dependent PI3K signaling14,15, mice with a knockout (KO) of the p85α regulatory subunit display increased insulin sensitivity, increased levels of PIP3 lipids, elevated Akt/PKB activity and improved glucose tolerance13,16–19. The molecular mechanisms that underlie this negative regulation by p85 appear to be complex and include unbalanced stoichiometry between subunits20,21; effects of p85 on both protecting p110 from degradation while partially inhibiting its kinase activity16,19,21,22; retention of PI3K in an inactive vesicle compartment23; links between p85α and PTEN activity24; links between p85α and JNK activity leading to IRS1 serine phosphorylation and inhibition of IRS1-mediated effects, and links between p85α and XBP-1 in modifying the unfolded protein response25.\n\nTo dissect the intricate equilibrium between the catalytic and regulatory subunits of PI3K, as well as the opposing and complex roles of p110α and p85α in insulin signaling and action, in the present study, we have investigated the impact of a combined hepatic deletion of p110α and p85α on insulin signaling and whole body glucose homeostasis.\n\n\nMethods\n\nAll mice in this study were on a 129Sv-C57Bl/6 mixed genetic background. To create the liver double knock-out mice, p110α lox-lox mice7 were crossed with p85α lox-lox mice, hemizygous for the Albumin-Cre recombinase transgene14. Mice were housed on a 12-hour light cycle and fed a standard rodent chow and water ad libitum. All protocols for animal use and euthanasia were approved by the Gothenburg Ethical Committee on Animal Experiments, in accordance with Swedish guidelines and Directive 2010/63/EU for animal experiments, and by the Animal Care Use Committee of the Joslin Diabetes Center and Harvard Medical School in accordance with National Institutes of Health guidelines. All efforts were made to ameliorate any suffering of the mice by reducing stress, hosting mice in small groups with items that stimulate their natural activity, and allowing the mice to recover 1–2 weeks after each procedure (such as measuring blood glucose, glucose tolerance test etc). During the insulin tolerance test, the mice were monitored closely to not fall too low in blood glucose levels.\n\nFor each experiment, a group of 5–12 male mice per genotype were used. The mice were studied from 6 weeks of age to 25 weeks of age. During this time weight and fasting blood glucose levels were measured every two weeks. Glucose tolerance test was performed at 8 weeks, 16 weeks and 24 weeks. Pyruvate tolerance test was performed at 15 weeks and insulin tolerance test was performed at 19 weeks.\n\nAnimals were fasted overnight and anesthetized with 2-2-2 tribromoethanol (Sigma-Aldrich, St Louis MO), followed by injection of 5 U of insulin (Actrapid, Novo Nordisk Inc., Plainsboro Township, NJ) or saline via the inferior vena cava. Five minutes after the injection, the liver, muscle and white adipose tissue (WAT) were excised, weighed, and snap-frozen in liquid nitrogen.\n\nGlucose tolerance test was performed by intraperitoneal (i.p.) injection of 2 g glucose/kg BW after an overnight fast. Insulin tolerance test was performed by i.p. injection of 1.25 U insulin/kg BW. Pyruvate tolerance test was performed by i.p. injection of 2 g of pyruvate/kg BW after an over-night fast. Insulin was measured with ELISA (Crystal Chem Inc., Downer Grover, IL).\n\nRNA was extracted by homogenization of liver tissue in RLT buffer (Qiagen, Valencia, CA) followed by extraction using the RNeasy kit (Qiagen, Valencia, CA). For gene analysis, cDNA was prepared using a high capacity cDNA archive kit (Applied Biosystems, Foster City, CA) with random hexamer primers. Gene expression was analyzed by real-time reverse transcription-PCR (RT-PCR) on an ABI Prism sequence detection system (Applied Biosystems, Foster City, CA). The cycling conditions used were an initial 95°C 10-minute step followed by 40 cycles of 95°C for 15s and 60°C for 60s. Samples were normalized to the 18S rRNA gene. Primer sequences are available in Table 1.\n\nLiver tissue was homogenized in lysis buffer containing 25 mM Tris-HCl, 2 mM Na3VO4, 10mM Na4P2O7, 1 mM EGTA, 1 mM EDTA, 1% NP-40 and protease inhibitors (Sigma-Aldrich, St Louis MO), then allowed to incubate at 4°C for one hour. Extracts were centrifuged at 55,000 rpm (Beckman 70.1 Ti rotor) for one hour, and the supernatant was stored at -80°C. WAT was homogenized in lysis buffer containing 25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 25 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 1 mM orthovanadate, and protease inhibitors (Sigma-Aldrich, St Louis MO) followed by incubation for 2 h at 4°C. The samples were then centrifuged at 12,000 rpm for 15 min, and the supernatant was collected and stored at -80°C. Protein analysis was made by SDS-PAGE and subsequent western blot. Briefly, protein samples were loaded onto 4–12% Bis-Tris protein gels (Thermo-Fisher Scientific, Waltham, MA) and subjected to gel electrophoresis using 25mM Tris, 192 mM glycine and 0.1% SDS as running buffer. Samples were transferred onto a nitrocellulose membrane and the membranes were incubated in 5% skim milk solution for 1 h followed by primary antibody incubation according to the manufacturer’s protocol for each antibody. Membranes were washed 2x5 min and 1x15 min in PBS with 0.1% Tween and then incubated with the secondary antibody for 1h followed by another washing procedure. Immunoprecipitation was performed using magnetic beads coated with protein G (Pierce Biotechnology Inc, Rockford, IL). All western blots and immunoprecipitation experiments were performed with a minimum of four replicates (four separate samples).\n\nIRS1 (RRID:AB_2127860, rabbit monoclonal, 1:50, Cell Signaling Technology Inc, cat# 3407 for western blot), IRS1 (RRID:AB_631842, rabbit polyclonal, 10μl per reaction, Santa Cruz Biotechnology Inc, cat# sc-559 for immunoprecipitation), p110α (rabbit monoclonal, 1:1000 for western blot and 1:50 for immunoprecipitation, Cell Signaling Technology Inc, cat# 4249), p110β (RRID:AB_2165246, rabbit monoclonal, 1:500 for western blot and 1:50 for immunoprecipitation, Cell Signaling Technology Inc, cat# 3011), p110γ (RRID:AB_10828316, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 5405), p110δ (mouse monoclonal, 1:500, Becton, Dickinson and Company, cat# 611015), p101 (RRID:AB_10829448, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 5569), Vps34 (RRID:AB_2299765, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 4263), p150 (rabbit polyclonal, 1:1000, Cell Signaling Technology Inc, cat# 14580), Akt/PKB (RRID:AB_329827, rabbit polyclonal, 1:1000, Cell Signaling Technology Inc, cat# 9272), pS-Akt/PKB (RRID:AB_329825, rabbit polyclonal, 1:1000, Cell Signaling Technology Inc, cat# 9271), pT-Akt/PKB (RRID:AB_2255933, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 2965), pT-p70S6K (RRID:AB_330944, rabbit polyclonal, 1:1000, Cell Signaling Technology Inc, cat# 9205), p85α (RRID:AB_2268174, rabbit monoclonal, 1:1000, Abcam, cat# 22653), p85-pan (RRID:AB_10831521, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 4257), pT202/Y204-ERK (RRID:AB_2315112, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 4370), ERK (RRID:AB_390779, rabbit monoclonal, 1:1000, Cell Signaling Technology Inc, cat# 4695), p55γ (mouse monoclonal, 1:2000, Abcam, cat# ab186612), PIK3C2α (rabbit polyclonal, 1:1000, MyBiosource, cat# MBS9202698), PIK3C2γ (rabbit polyclonal, 1:1000, MyBiosource, cat# MBS820611). Rabbit secondary antibody (RRID:AB_772206, HRP-linked from donkey, 1:1000, GE Healthcare Life Sciences, cat# NA934), mouse secondary antibody (RRID:AB_772210, HRP-linked from sheep, 1:1000, GE Healthcare Life Sciences, cat# NA931).\n\nImmunoprecipitates, using protein G Dynabeads (Life Technologies), of liver protein lysates were prepared with p110α and p110β antibodies or IRS1 antibody. The PI3K assay was performed as previously described13. Briefly, immunoprecipitates were incubated with 5 μg PI substrate (phosphatidylinositol from bovine liver), 20 mM MgCl2, 8 μM cold ATP and 0.5 μl radio-labeled [γ-32P]-ATP (1.111014 bq/mmol) in PI3K reaction buffer (20 mM Tris-HCl, 100 mM NaCl and 0.5 mM EGTA) for 25 min at room temperature. The resulting radioactively labeled PIP was analyzed with thin layer chromatography and phosphorimaging (FLA-3000, Fujifilm). Prior to these experiments, titration experiments of bead concentration and PI substrate concentration were performed to ensure precipitation of equal amounts of protein, as well as optimal PI concentration to obtain maximal enzyme activity.\n\nAll data are presented as mean ± standard error of the mean (SEM). Student's t-test was used for statistical analysis between two unpaired groups. A p-value of <0.05 was considered statistically significant. The statistical software used was GraphPad Prism 7.00.\n\n\nResults\n\nMice with a liver-specific deletion of p110α and p85α, termed hereafter liver double knockout (L-DKO) mice, were created by breeding mice carrying homozygous floxed Pik3ca and Pik3r1 alleles14,26 with transgenic mice carrying the Cre recombinase driven by the albumin promoter (albumin-Cre). Deletion of Pik3caand Pik3r1 in the liver resulted in markedly reduced gene and protein expression of p110α and p85α (Figures 1A, 1C, 1E), as well as impaired activation of the downstream targets Akt/PKB, with decreased phosphorylation of serine 473 and threonine 308, and p70S6 kinase (Figure 1F). p110β gene expression was not affected by the deletion of p110α and p85α (Figure 1B). p85β gene expression was slightly, but significantly, decreased in the L-DKO livers (Figure 1D). As expected, in the floxed control mice, there was an increase in the amount of p110α associated with IRS1 in response to insulin compared to basal conditions, whereas no p110α was associated with IRS1 in the L-DKO mice (Figure 1G). MAPK signaling, as shown by ERK phosphorylation, was unchanged in the L-DKO mice compared to controls (Figure 1F).\n\nmRNA expression of (A) Pik3ca, (B) Pik3cb, (C) Pik3r1 and (D) Pik3r2 in livers of flox controls and L-DKO mice. (E) Representative western blot of protein expression of p110α, p110β, p85α, p85-pan (detects both p85 isoforms) and p55γ and (F, phosphorylated Akt/PKB (Ser 473 and Thr 308), phosphorylated p70S6 kinase, and phosphorylated ERK in livers of flox controls and L-DKO mice. Total ERK was used as a loading control. (G) Representative western blot of immunoprecipitation experiments with antibodies for IRS1 and subsequent immunoblotting with antibodies for p110α, p110β, p110δ, p85α, p85-pan (detects both p85 isoforms) and p55γ. The whole-lysate reference sample was from an insulin-treated flox control mouse. IP = immunoprecipitation, IB = immunoblot. Basal condition is indicated with a minus (-) sign, insulin-treated condition is indicated with a plus (+) sign. Basal conditions refer to fasting of mice overnight. Insulin treatment refers to injection of 5 U of insulin through the vena cava 5 min prior to euthanization. Error bars indicate SEM (n = 5–8). , p < 0.05 compared to controls. n.s = non significant. Images containing a vertical line are composites taken from a single original image.\n\nPrevious studies have shown that the interaction between the regulatory and catalytic subunits of PI3K to form dimers has a mutual stabilizing effect on both subunits, whereas the monomeric forms are more readily subjected to degradation19,21,22. We hypothesized that more p85β would bind to IRS1 when p85α was absent, thereby maintaining p110β stabilization. However, only very low levels of p85β protein were detected in the liver of the L-DKO mice, as shown both in assessment of total p85 protein (Figure 1E) and in p85 immunoprecipitates with IRS1 (Figure 1G). The expression of p55γ regulatory isoform protein was not affected by deletion of p110α and p85α (Figure 1E), nor was the amount bound to IRS1 (Figure 1G). In contrast, total p110β protein expression was decreased in the L-DKO mice compared to controls (Figure 1E), likely due to destabilization of this catalytic isoform in the absence of p85α, supporting an insufficiency for p85β to compensate for the loss of p85α. Interestingly, despite overall decreased protein expression of p110β, similar amounts of p110β were associated with IRS1 in controls and L-DKO mice (Figure 1G). The third catalytic isoform of class IA PI3Ks, p110δ, has been shown to have a major role in immune cells and the embryonic nervous system, but not in other tissues10–12. Consistent with this, we found only very small amounts of full length p110δ in whole liver lysates or bound to IRS1 (Figure 1G). However, the antibody picked up a band of about 70 kDa in whole lysates and in IRS1 immunoprecipitates (Figure 1G). The amount of this protein did not appear to be consistently different between controls and L-DKO mice or between basal and insulin-stimulated samples, so is likely non-specific.\n\nAs expected, p110α kinase activity, as assessed by the ability to add the 3’-phosphate group to phosphoinositides in p110α immunoprecipitates, was markedly decreased both in the basal- and insulin-stimulated states of the L-DKO mice compared to controls (Figure 2A). Overall p110β kinase activity was much lower than p110α kinase activity in control mice, consistent with previous studies7,27, and was not changed in the basal state between controls and L-DKO mice (Figure 2B). However, p110β kinase activity was significantly decreased in the insulin-stimulated state of the L-DKO mice (Figure 2B), even though similar amounts of p110β protein were associated with IRS1 in controls and L-DKO mice (Figure 1G). Surprisingly, IRS1-associated phosphatidylinositol kinase activity in response to insulin was intact in the L-DKO, despite lack of p110α kinase activity and decreased p110β activity (Figure 2C).\n\n(A) p110α lipid kinase activity, (B) p110β lipid kinase activity, and (C) IRS1-associated phosphatidylinositol (PI) kinase activity in L-DKO mice and controls were assessed by immunoprecipitation. (D) Representative western blot of immunoprecipitation experiments with antibodies for IRS1 and subsequent immunoblotting with antibodies for p110γ, p101, Vps34, p150, PIK3-C2α and PIK3-C2γ. The whole-lysate reference sample was from an insulin-treated flox control mouse. IP = immunoprecipitation, IB = immunoblot. Basal condition is indicated with a minus (-) sign, insulin-treated condition is indicated with a plus (+) sign. Basal conditions refer to fasting of mice overnight. Insulin treatment refers to injection of 5 U of insulin through the vena cava 5 min prior to euthanization. Error bars indicate SEM (n = 4). , p < 0.05 compared to controls.\n\nTo explore if other classes of phosphoinositide 3-kinases were responsible for the intact IRS1-associated activity, we investigated the association of IRS1 with class IB-, class II and class III members of phosphoinositide 3-kinases in liver lysates from controls and L-DKO mice. Class IB PI3K consists of the p110γ catalytic subunit and the p101 regulatory subunit. The amount of p101 was low in whole lysates and no p101 was associated with IRS1 in either control mice or L-DKO mice (Figure 2D). p110γ was associated with IRS1, but the amounts were similar between controls and L-DKO mice (Figure 2D). Class III PI3K catalytic subunit Vps34 and regulatory subunit p150 were present in whole lysates, but not associated with IRS1 in either control mice or L-DKO mice (Figure 2D). Class II PI3K consists of only one subunit, but exists in several isoforms. The PI3K-C2α isoform is ubiquitously expressed, whereas PI3K-C2γ has been reported to have a more limited tissue distribution, including hepatocytes28,29. However, we detected only little or no PI3K-C2α or PI3K-C2γ in whole liver lysates or associated with IRS1 in controls or L-DKO mice (Figure 2D). Thus, other classes of phosphoinositide 3-kinases did not compensate for the loss of p110α and p85α in the L-DKO mice and could not explain the intact IRS1-associated phosphatidylinositol kinase activity in the absence of p110α and p85α.\n\nOn standard chow (4% fat content by weight, 12% by calories), L-DKO mice had similar body weights to the flox control mice until 10–12 weeks of age, after which the L-DKO mice showed slower weight gain compared to controls (Figure 3A). At least part of this decrease was due to a decrease in liver weight. Thus, by 10 weeks of age, the ratio of liver weight to body weight in the L-DKO mice was decreased by 13% compared to control mice (Figure 3B), whereas there was no difference in WAT weight or muscle weight (Figures 3C and 3D). At 24 weeks of age, liver weight remained decreased (18%), and there was also a significant decreased WAT weight (22%) for the L-DKO mice compared to controls (Figures 3B and 3C). To assess a possible change in insulin sensitivity in WAT, associated with the decreased WAT mass, we investigated insulin signaling mediators in this tissue. WAT p110α- and p85α expression was similar in controls and L-DKO mice as was Akt/PKB phosphorylation in response to insulin (Figure 3E). There was no difference in insulin-stimulated Akt/PKB-activation at 10w compared to 25w in either controls or L-DKO mice (Figure 3E).\n\n(A) whole body weight, (B) liver weight, (C) white adipose tissue (WAT) weight and (D) muscle weight of 10 and 24 week old male L-DKO mice and controls. (E) Representative western blot of p110α, p85α and phosphorylated Akt/PKB (Ser 473) in WAT of 10 and 25 week old male flox controls and L-DKO mice. Total Akt/PKB was used as a loading control. Basal condition is indicated with a minus (-) sign, insulin-treated condition is indicated with a plus (+) sign. Basal conditions refer to fasting of mice overnight. Insulin treatment refers to injection of 5 U of insulin through the vena cava 5 min prior to euthanization. Error bars indicate SEM (n = 8–17). , p < 0.05 compared to controls. w = weeks.\n\nDespite similar body weight (Figure 3A), L-DKO mice were severely glucose intolerant as early as at 8 weeks of age (Figure 4A), and remained similarly glucose intolerant throughout the 24 week study (Figures 4B and 4C). This was associated with markedly increased fasting insulin levels (Figure 4D). However, somewhat surprisingly, the L-DKO mice showed a normal response to exogenous insulin during an intraperitoneal insulin tolerance test (Figure 4E). Despite the markedly impaired glucose tolerance and marked hyperinsulinemia, fasting and random fed glucose levels in the L-DKO mice remained similar between controls and L-DKO mice (Figures 4F and 4G).\n\nGlucose tolerance test at (A) 8 weeks, (B) 16 weeks and (C) 24 weeks of age; (D) fasting insulin levels at 10 weeks of age; (E) insulin tolerance test at 19 weeks of age; (F) fasting glucose levels; (G) random fed glucose levels; (H) pyruvate tolerance test at 15 weeks of age; (I–K) hepatic mRNA expression of the gluconeogenic markers glucose 6-phosphatase (G6pc), phosphoenolpyruvate carboxykinase (Pck1) or fructose 1,6-bisphosphatase (Fbp1). L-DKO mice and controls were given 2 g glucose/kg body weight intraperitoneally (i.p.) for the glucose tolerance test or 1.25 U insulin/kg body weight i.p. for the insulin tolerance test or 2 g pyruvate/kg body weight i.p. for the pyruvate tolerance test. Blood glucose was measured at 0, 15, 30, 60, and 120 min. Error bars indicate SEM (n =6–12). *, p < 0.05. The insets in the glucose tolerance test graphs and the pyruvate tolerance test graph show the area under the curve (AUC) with subtracted basal glucose values.\n\nIncreased gluconeogenesis is one of the hallmarks of hepatic insulin resistance in type 2 diabetes. To determine whether the impaired glucose tolerance in L-DKO mice was due to increased hepatic glucose output, we subjected these animals to a challenge with pyruvate, the major gluconeogenic substrate. Over the 120 min period following administration of pyruvate, there was a trend toward increased glucose levels in L-DKO mice compared to controls, but this was not statistically significant (Figure 4H). This was associated with a significant change in hepatic gene expression of the gluconeogenic enzyme glucose 6-phosphatase (G6pc) (Figure 4I), whereas gene expression of the other key mediators of gluconeogenesis, phosphoenolpyruvate carboxykinase (Pck1) and fructose 1,6-bisphosphatase (Fbp1), remained similar between control mice and L-DKO mice (Figures 4J and 4K).\n\n\nDiscussion\n\nIn this study, we investigated the impact of a combined deletion of p110α and p85α on insulin signaling and glucose homeostasis. For this purpose, we created mice with a liver-specific deletion of the major catalytic and major regulatory subunits of PI3K: Pik3ca and Pik3r1. We have previously shown that hepatic deletion of only p110α results in severe insulin resistance and impaired glucose tolerance, signifying that p110α is crucial for mediating insulin signaling7. Moreover, mice deficient in all p85 isoforms in either muscle or liver exhibit severely impaired insulin signaling in these tissues14,15. The liver plays a crucial role in maintaining glucose homeostasis; we therefore hypothesized that deleting both these isoforms would result in severe and overt diabetes.\n\nAs expected, in the L-DKO mice, p110α catalytic activity was blunted and, as a result, the activation of the signal downstream of PI3K was markedly decreased. However, the L-DKO mice showed normal fasting and fed blood glucose levels throughout the study (24 weeks) and normal insulin tolerance. Glucose tolerance was impaired in the L-DKO mice and circulating insulin levels were markedly elevated, but to a degree similar to mice with only p110α deleted in the liver (L-p110α KO)7. Surprisingly, despite abolished p110α activity, we observed an intact total IRS1-associated phosphatidylinositol (PI) kinase activity in the L-DKO mice. This finding was very different from what we previously observed in L-p110α KO mice, which only have p110α deleted in the liver7. In the L-p110α KO mice, insulin-stimulated IRS1-associated PI kinase activity was markedly blunted7. This suggests that it is the loss of the regulatory subunit that accounted for the preserved IRS1-associated activation, possibly by enabling other phosphatidylinositol kinases to bind to IRS1. However, there did not appear to be any compensatory effects of other known catalytic and regulatory class IA subunit isoforms for the action of insulin. Thus, we did not detect any differences in the IRS1-associated amounts of p110β, p110δ or p55γ in controls and L-DKO mice in response to insulin and only very small amounts of p85β associated with IRS1 in the L-DKO mice. Similarly, no evidence of compensatory effects by class IB, class II or class III members of phosphoinositide 3-kinases were found.\n\nPrevious studies by us and others, including our study of the L-p110α KO mice, have shown that p110β is unable to compensate for the loss of p110α7,26,30. However, we observed similar amounts of IRS1-associated p110β in the L-DKO mice and controls despite overall total decreased levels of p110β in the liver. We therefore speculated that perhaps p110β activity was increased in response to insulin in the L-DKO mice compared to controls, which would explain the sustained IRS1-associated PI kinase activity. We found no difference in the p110β activity between the controls and the L-DKO mice in the basal state. In addition, the p110β kinase activity was significantly decreased, rather than increased, in the insulin-stimulated state of L-DKO mice. We thus conclude that the sustained IRS1-associated PI kinase activity in the L-DKO mice is not due to increased activity of p110β.\n\nAlthough the amount of IRS1-associated p110β was similar in controls and L-DKO, the total amount of p110β was decreased in the L-DKO mice. We, and others, have previously reported that the dimeric interaction between the regulatory and the catalytic subunits results in stabilization of the subunits, whereas the monomeric forms are more readily subjected to degradation18,20–21. Therefore, the absence of the major regulatory subunit p85α in the L-DKO mice probably subjects p110β to more rapid degradation. In this context, observing similar amounts of p110β associated with IRS1 in controls and L-DKO mice is somewhat surprising, but is likely due to stabilization of a fraction of p110β subunits by interaction with IRS1-associated p85β or p55γ.\n\nLack of the major regulatory subunit in the L-DKO mice, accompanied by absence of increased activation of p110β or compensatory increased expression of other phosphoinositide 3-kinases, suggests that presence of other classes of phosphatidylinositol kinases, perhaps PI4K and PI5K, account for the intact IRS1-associated kinase activity by directly binding to IRS1. PI4Ks have been described as mediators of endosomal trafficking from the Golgi and to be involved in EGF-stimulated phosphoinositide signaling31. Type II PI4Ks interact with the EGF receptor, but they are not known to interact with IRS1. Type III PI4Ks are structurally related to PI3Ks, with a high degree of conservation between their catalytic domains and sensitivity to wortmannin31. The isoform PI4KIIIα has been reported to be functionally connected to PI3K in FGF signaling during pectoral fin development in the zebra fish32.\n\nPI5K exists as two separate classes, PI(3)P5K and PI(4)P5K, phosphorylating the D5 position of the inositol ring of phosphatidylinositol 3-phosphate and phosphatidylinositol 4-phosphate, respectively. Of the PI(4)P5Ks, the isozyme PIP5Kc has been shown to respond to, and become phosphorylated by, various hormones and growth factors, such as EGF, and play a role in actin cytoskeletal reorganization, clathrin-dependent endocytosis, membrane ruffle formation, etc.33. However, a direct effect on insulin signaling and interaction with IRSs by PIP5Kc has not been reported. PI(3)P5K, also called PIKfyve, and has been quite extensively studied and reported to be involved in membrane trafficking, stress- or hormone-induced signaling, ion channel activity, cytoskeletal dynamics, nuclear transport, gene transcription and cell cycle progression34. Interestingly, PIKfyve is regulated by insulin, recruiting PIKfyve to inner membranes, where insulin receptor and IRSs are also found35, and co-precipitates with p110 and p85 subunits in 3T3-L1 adipocytes36. Thus, PIKfyve appears to be a possible contributor to the sustained IRS1-associated kinase activity in the L-DKO mice. However, PIKfyve expression has been reported to be rather tissue specific, mainly expressed in adipose tissue, muscle and brain37,38 and expression in the liver appears low37,38. A more extensive follow-up investigation of the various phosphoinositides in the L-DKO livers compared to controls may help elucidating the phosphatidylinositol kinase responsible for the sustained IRS1-associated kinase activity in the L-DKO mice.\n\nThe absence of both p85α and p110α, as seen in the L-DKO mice, would logically result in a very severely impaired metabolic phenotype. The L-DKO mice had markedly elevated circulating insulin levels, impaired glucose tolerance and showed a trend toward increased rates of hepatic glucose output when given pyruvate. However, fasted and fed glucose levels were not different between controls and L-DKO mice, i.e., randomly fed L-DKO mice were not diabetic and insulin tolerance tests were normal. Part of this protection might be the fact that L-DKO mice had less accumulation of WAT with age. In addition, it is possible that the high circulating insulin levels reflect an impaired hepatic insulin clearance rather than insulin resistance in the muscle, which would explain the paradoxical normal insulin tolerance. Interestingly, body weight, WAT weight and hepatic glucose output were significantly increased and insulin tolerance severely impaired in L-p110α KO mice7, which lack only p110α in liver, demonstrating that L-DKO mice showed an overall less severe metabolic phenotype compared to L-p110α KO mice.\n\nIn summary, deletion of hepatic p110α and p85α results in an impaired insulin signal and impaired glucose homeostasis, but shows an overall less severe metabolic phenotype compared to mice with only p110α deleted in the liver. Although other PI3Ks were unable to compensate for the loss of p110α and p85α, IRS1-associated phosphatidylinositol kinase activity was surprisingly still intact, possibly due to interaction of IRS1 with other classes of phosphatidylinositol kinases.\n\nAbbreviations: Fbp, fructose-1,6-bisphosphatase; G6pc, glucose-6-phosphatase; GTT, glucose tolerance test; ITT, insulin tolerance test; L-DKO, liver double knockout; Pck, phosphoenolpyruvate carboxykinase; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PKB, protein kinase B; PTT, pyruvate tolerance test; WAT, white adipose tissue\n\n\nData availability\n\nDataset 1: Raw data for gene and protein expression shown in Figure 1. DOI, 10.5256/f1000research.12418.d17549339\n\nDataset 2: Raw data for PI3K activity measurements and protein expression shown in Figure 2. DOI, 10.5256/f1000research.12418.d17549440\n\nDataset 3: Raw data for body and tissue weights and protein expression shown in Figure 3. DOI, 10.5256/f1000research.12418.d17549541\n\nDataset 4: Raw data for metabolic procedures and measurements and gene expression shown in Figure 4. DOI, 10.5256/f1000research.12418.d17549642", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Swedish Research Council (VRS), the Sahlgrenska Academy Foundation (VRS), the Magnus Bergvall foundation (VRS), and the Wilhelm and Martina Lundgren Foundation (VRS). The work was also supported by NIH grant DK055545 (CRK).\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\nEngelman JA, Luo J, Cantley LC: The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet. 2006; 7(8): 606–619. PubMed Abstract \| Publisher Full Text\n\nTaniguchi CM, Emanuelli B, Kahn CR: Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol. 2006; 7(2): 85–96. PubMed Abstract \| Publisher Full Text\n\nFransson S, Uv A, Eriksson H, et al.: p37δ is a new isoform of PI3K p110δ that increases cell proliferation and is overexpressed in tumors. Oncogene. 2012; 31(27): 3277–3286. PubMed Abstract \| Publisher Full Text\n\nEjeskär K, Vickes O, Kuchipudi A, et al.: The Unique Non-Catalytic C-Terminus of P37delta-PI3K Adds Proliferative Properties In Vitro and In Vivo. PLoS One. 2015; 10(5): e0127497. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFoukas LC, Claret M, Pearce W, et al.: Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation. Nature. 2006; 441(7091): 366–370. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatton DT, Garçon F, Okkenhaug K: The PI3K p110delta controls T-cell development, differentiation and regulation. Biochem Soc Trans. 2007; 35(Pt 2): 167–171. PubMed Abstract \| Publisher Full Text\n\nBilancio A, Okkenhaug K, Camps M, et al.: Key role of the p110delta isoform of PI3K in B-cell antigen and IL-4 receptor signaling: comparative analysis of genetic and pharmacologic interference with p110delta function in B cells. Blood. 2006; 107(2): 642–650. PubMed Abstract \| Publisher Full Text\n\nEickholt BJ, Ahmed AI, Davies M, et al.: Control of axonal growth and regeneration of sensory neurons by the p110delta PI 3-kinase. PLoS One. 2007; 2(9): e869. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUeki K, Algenstaedt P, Mauvais-Jarvis F, et al.: Positive and negative regulation of phosphoinositide 3-kinase-dependent signaling pathways by three different gene products of the p85alpha regulatory subunit. Mol Cell Biol. 2000; 20(21): 8035–8046. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaniguchi CM, Kondo T, Sajan M, et al.: Divergent regulation of hepatic glucose and lipid metabolism by phosphoinositide 3-kinase via Akt and PKClambda/zeta. Cell Metab. 2006; 3(5): 343–353. PubMed Abstract \| Publisher Full Text\n\nLuo J, Sobkiw CL, Hirshman MF, et al.: Loss of class IA PI3K signaling in muscle leads to impaired muscle growth, insulin response, and hyperlipidemia. Cell Metab. 2006; 3(5): 355–366. PubMed Abstract \| Publisher Full Text\n\nFruman DA, Mauvais-Jarvis F, Pollard DA, et al.: Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha. Nat Genet. 2000; 26(3): 379–382. PubMed Abstract \| Publisher Full Text\n\nMauvais-Jarvis F, Ueki K, Fruman DA, et al.: Reduced expression of the murine p85alpha subunit of phosphoinositide 3-kinase improves insulin signaling and ameliorates diabetes. J Clin Invest. 2002; 109(1): 141–149. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTerauchi Y, Tsuji Y, Satoh S, et al.: Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase. Nat Genet. 1999; 21(2): 230–235. PubMed Abstract \| Publisher Full Text\n\nBrachmann SM, Ueki K, Engelman JA, et al.: Phosphoinositide 3-kinase catalytic subunit deletion and regulatory subunit deletion have opposite effects on insulin sensitivity in mice. Mol Cell Biol. 2005; 25(5): 1596–1607. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarbour LA, Mizanoor Rahman S, Gurevich I, et al.: Increased P85alpha is a potent negative regulator of skeletal muscle insulin signaling and induces in vivo insulin resistance associated with growth hormone excess. J Biol Chem. 2005; 280(45): 37489–37494. PubMed Abstract \| Publisher Full Text\n\nUeki K, Fruman DA, Brachmann SM, et al.: Molecular balance between the regulatory and catalytic subunits of phosphoinositide 3-kinase regulates cell signaling and survival. Mol Cell Biol. 2002; 22(3): 965–977. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu J, Zhang Y, McIlroy J, et al.: Regulation of the p85/p110 phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit. Mol Cell Biol. 1998; 18(3): 1379–1387. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo J, Field SJ, Lee JY, et al.: The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex. J Cell Biol. 2005; 170(3): 455–464. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaniguchi CM, Tran TT, Kondo T, et al.: Phosphoinositide 3-kinase regulatory subunit p85alpha suppresses insulin action via positive regulation of PTEN. Proc Natl Acad Sci U S A. 2006; 103(32): 12093–12097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaniguchi CM, Aleman JO, Ueki K, et al.: The p85alpha regulatory subunit of phosphoinositide 3-kinase potentiates c-Jun N-terminal kinase-mediated insulin resistance. Mol Cell Biol. 2007; 27(8): 2830–2840. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao JJ, Cheng H, Jia S, et al.: The p110alpha isoform of PI3K is essential for proper growth factor signaling and oncogenic transformation. Proc Natl Acad Sci U S A. 2006; 103(44): 16296–16300. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaudhari A, Krumlinde D, Lundqvist A, et al.: p110α Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110α Kinase Activity. Mol Cell Biol. 2015; 35(19): 3258–3273. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDomin J, Pages F, Volinia S, et al.: Cloning of a human phosphoinositide 3-kinase with a C2 domain that displays reduced sensitivity to the inhibitor wortmannin. Biochem J. 1997; 326(Pt 1): 139–147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRozycka M, Lu YJ, Brown RA, et al.: cDNA cloning of a third human C2-domain-containing class II phosphoinositide 3-kinase, PI3K-C2gamma, and chromosomal assignment of this gene (PIK3C2G) to 12p12. Genomics. 1998; 54(3): 569–574. PubMed Abstract \| Publisher Full Text\n\nBi L, Okabe I, Bernard DJ, et al.: Proliferative defect and embryonic lethality in mice homozygous for a deletion in the p110alpha subunit of phosphoinositide 3-kinase. J Biol Chem. 1999; 274(16): 10963–10968. PubMed Abstract \| Publisher Full Text\n\nBalla A, Balla T: Phosphatidylinositol 4-kinases: old enzymes with emerging functions. Trends Cell Biol. 2006; 16(7): 351–361. PubMed Abstract \| Publisher Full Text\n\nMa H, Blake T, Chitnis A, et al.: Crucial role of phosphatidylinositol 4-kinase IIIalpha in development of zebrafish pectoral fin is linked to phosphoinositide 3-kinase and FGF signaling. J Cell Sci. 2009; 122(Pt 23): 4303–4310. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFunakoshi Y, Hasegawa H, Kanaho Y: Regulation of PIP5K activity by Arf6 and its physiological significance. J Cell Physiol. 2011; 226(4): 888–895. PubMed Abstract \| Publisher Full Text\n\nJin N, Lang MJ, Weisman LS: Phosphatidylinositol 3,5-bisphosphate: regulation of cellular events in space and time. Biochem Soc Trans. 2016; 44(1): 177–184. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShisheva A, Rusin B, Ikonomov OC, et al.: Localization and insulin-regulated relocation of phosphoinositide 5-kinase PIKfyve in 3T3-L1 adipocytes. J Biol Chem. 2001; 276(15): 11859–11869. PubMed Abstract \| Publisher Full Text\n\nSbrissa D, Ikonomov O, Shisheva A: Selective insulin-induced activation of class IA phosphoinositide 3-kinase in PIKfyve immune complexes from 3T3-L1 adipocytes. Mol Cell Endocrinol. 2001; 181(1–2): 35–46. PubMed Abstract \| Publisher Full Text\n\nShisheva A, Sbrissa D, Ikonomov O: Cloning, characterization, and expression of a novel Zn2+-binding FYVE finger-containing phosphoinositide kinase in insulin-sensitive cells. Mol Cell Biol. 1999; 19(1): 623–634. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIkonomov OC, Sbrissa D, Delvecchio K, et al.: Muscle-specific Pikfyve gene disruption causes glucose intolerance, insulin resistance, adiposity, and hyperinsulinemia but not muscle fiber-type switching. Am J Physiol Endocrinol Metab. 2013; 305(1): E119–131. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaudhari A, Ejeskär K, Wettergren Y, et al.: Dataset 1 in: Hepatic deletion of p110a and p85a results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity. F1000Research. 2017. Data Source\n\nChaudhari A, Ejeskär K, Wettergren Y, et al.: Dataset 2 in: Hepatic deletion of p110a and p85a results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity. F1000Research. 2017. Data Source\n\nChaudhari A, Ejeskär K, Wettergren Y, et al.: Dataset 3 in: Hepatic deletion of p110a and p85a results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity. F1000Research. 2017. Data Source\n\nChaudhari A, Ejeskär K, Wettergren Y, et al.: Dataset 4 in: Hepatic deletion of p110a and p85a results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity. F1000Research. 2017. Data Source" }	[ { "id": "25513", "date": "14 Sep 2017", "name": "James R. Woodgett", "expertise": [ "Reviewer Expertise Signal transduction" ], "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 study, the authors generated a liver-specific double knockout of the regulatory and catalytic subunit of Class 1A PI3K. Previously, single inactivation of either the catalytic art regulatory subunits had been shown to result in opposite effects on insulin resistance vs sensitivity. The authors demonstrated through in vivo studies in male mice (129Sv-C57Bl/6) that defects in glucose tolerance as well as changes in liver, fat and body weight occur upon deletion of both the regulatory PIK3R1(p85α) and catalytic subunit PIK3CA (p110α) but retained normal production of gulches from the liver.\nSpecific Comments:\nFigure 1E (page 6): the authors state (on page 5) that low levels of p85β were detected in the L-DKO mice compared to controls. This was indirectly demonstrated through the use of a pan-p85 antibody that detects both p85α and p85β. Mice with liver-specific deletion of p85α on a p85β null background have been previously characterized (Taniguchi et al.). There, the overall levels of p85β detected utilizing a pan p85β antibody in a lysate derived from a liver that was wild type for p85β and null for p85α was minimal. Therefore, it is possible that the overall protein content reduction observed using the pan p85 antibody in Figure1E may be explained by the deletion of p85α from the liver, and may not be due to any change in p85β levels. To directly compare wild type and L-DKO samples, a specific p85β antibody should be used. Loading controls should also be provided for some panels in Figure 1.\n\nFigure 1F to show impaired activation, total AKT blots should be provided (similar to Figure 3E). Total levels of p70S6K also need to be measured. In Figure 1G, the authors state that although p110β levels are reduced, the amount associated with IRS-1 is unchanged. However, due to the variability across samples in both control and L-DKO, and the overall poor quality of the blot, these conclusions don’t inspire confidence. These data should be quantitated. Additionally, the p110β band on the last lane appears to run slightly higher compared to the rest of the bands and the control raising possible questions over the specificity of the p110β antibody used for IP. Are similar results observed using a distinct antibody? In line with the comments in point 1, the authors state that the overall amount of p85β associated IRS-1 does not change. This conclusion requires use of a p85β selective antibody.\n\nAs noted by the authors, there is known complexity between the structural roles of the various PI3K subunits and isoforms, with effects on protein stability and access to IRS-1 binding sites. There appears, as noted above, to be significant variation in levels of the untargetted subunits across the 2 typically shown animals (e.g. Figure 1G). If other animals were measured, a composite of the levels (e.g. n=4) would provide much better insight into the levels (if any) of compensation, allowing a clearer picture of what may be underlying the glucose metabolic effects. It is also possible there was downstream adaptation given the IRS-1 is negatively regulated by PI3K signaling (and appears somewhat higher in the DKOs (Figure 1G).\n\nFor the PI3K lipid kinase activity measurements (chromatographic separation, Figure 2 shows the quantitation), how were the separate plates normalized?\n\nMinor grammatical error: Page 11, second paragraph line 10- “and” is added in error.\n\n*Taniguchi, C. M., T. Kondo, M. Sajan, J. Luo, R. Bronson, T. Asano, R. Farese, L. C. Cantley and C. R. Kahn (2006). \"Divergent regulation of hepatic glucose and lipid metabolism by phosphoinositide 3-kinase via Akt and PKClambda/zeta.\" Cell Metab 3(5): 343-353.\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? Yes", "responses": [ { "c_id": "3662", "date": "05 Jun 2018", "name": "Victoria Rotter Sopasakis", "role": "Author Response", "response": "1. We totally agree with the Dr Woodgett’s point here and it is essentially what we tried to convey in the manuscript, but to make it clearer we have added an extra sentence including the reference to Taniguchi et al (page 9, row 7). We have previously (before submitting the manuscript) tried various different p85b antibodies without any success. Some work well for human tumor tissue for example, but give no band at all for our mouse liver lysates. Other antibodies we tried fail to deliver a band for both human and mouse tissues. This is the reason we chose to use the p85-pan antibody to indirectly show p85b protein expression. Since both Dr Woodgett and Drs Lin and Ballou requested a p85b antibody we have tried again to make various p85b antibodies on the market to work for our liver lysates and have spent months now trying to optimize the western blot conditions for these antibodies. We are not satisfied with the results. The only conclusion we can draw is that either 1) the p85b antibodies available on the market simply does not work well for mouse tissue or 2) there is not much p85b expressed in mouse liver (just as Dr Woodgett points out above). Based on the p85-pan westerns that we performed as well as previous studies including Taniguchi et al, to which Dr Woodgett referred, we believe that there simply is not that much p85b expressed in mouse liver. Loading controls have been added to figure 1 E and 1F, including Akt and p70S6K. 2. Total Akt blot has been added to figure 1F Total p70S6K has been added to figure 1F Blots in figure 1G (=new figure 1K) have been quantitated, except for p110d, which is so weak there is no point to quantify it. Graphs of the quantitation have been added to figure 1 (new figures 1L-P). n=4 in these quantification graphs, i.e we have added more samples for each condition to cover small variations in the immunoprecipitation and western blot experiments. p85b has not been added for the same reason as in figure 1E, see above. 3. In the quantification graphs added to figure 1F (new figure 1G-J) n=4, ie we have added new experiments with different samples. The WB pictures become very large when adding so many panels, so we decided including all four replicates in the quantification graphs. However, all blots are provided in the raw data sets for the referee to see. Same for figure 1G (new figure 1K), i.e n=4. Quantification graphs have been added (new figures 1L-P). 4. The plates were normalized to a reference sample = active p110a/p85 complex (see original plate pictures in the raw data sets). 5. Can not find this error. Please specify." } ] }, { "id": "27376", "date": "08 Nov 2017", "name": "Lily Q. Dong", "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\nChaudhari et al. reported the physiologic roles of hepatic p110a and p85a in regulating insulin signaling and glucose homeostasis. They concluded that hepatic deletion of p110α and p85α (L-DKO) led to mild metabolic phenotype of the L-DKO mice and proposed that lipid kinases other than PI3Ks might compensate for the loss of p110α/p85α by signaling through unknown signaling pathway other than Akt signaling. Since deletion of p85α leads to improved insulin sensitivity and deletion of p110α leads to impaired insulin signaling and insulin resistance, it is interesting to learn that L-DKO mice showed an overall less severe metabolic phenotype compared to the L-p110a KO mice.\n\nAlthough the expression levels of p110b were decreased in the liver of L-DKO mice (Fig. 1E), IRS1-associated p110b levels were comparable between the control and KO mice (Fig. 1G). In addition, notable levels of p110b activity were maintained in the L-DKO mice and insulin treatment can stimulate the activity although it is 50% compared to the control (Fig. 2B). It is unclear why the authors concluded that the intact IRS1-associated kinase activity in the KO mice are not from the remaining p110b.\n\nThe KO mice showed significantly impaired glucose tolerance and markedly increased fasting insulin levels (Fig. 4). What were the pancreatic phenotype of the KO mice? What were the levels of glucagon in the KO mice?\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": "3663", "date": "05 Jun 2018", "name": "Victoria Rotter Sopasakis", "role": "Author Response", "response": "1. We don’t believe that the intact IRS1-associated kinase activity in the DKO mice are from the remaining p110b for the following reasons: The total degree of kinase activity is very much smaller for p110b compared to p110a. This is an observation we have made over and over again for liver lysates in response to insulin in several different studies (the a.u numbers of the y-axis in the graphs in figure 2 demonstrates this to some extent). However, we still wanted to be sure that we were correct to rule out a role of p110b since we observed similar amounts of p110b bound to IRS1 in the DKO and controls. It was therefore important to look at the kinase activity associated with IRS1. The p110b-associated kinase activity in response to insulin was significantly decreased in the DKO mice compared to controls (figure 2b). These two observations ( 1) very much smaller degree of p110b activity over all compared to p110a kinase activity and 2) decreased IRS1-associated p110b activity in the DKO in response to insulin) makes it highly inlikely that the sustained IRS1-associated kinase activity is due to compensatory levels of p110b kinase activity). 2. We have performed a glucagon ELISA assay for the mice (new figure 4H). There were no differences in glucagon serum levels between DKO mice and controls at either 10 weeks of age or 24 weeks of age. The high fasting insulin levels in the DKO mice suggest that insulin production and secretion by the pancreas is intact and since no differences in glucagon secretion was observed even at older age we conclude that the pancreatic phenotype is likely very similar between DKO mice and controls." } ] }, { "id": "27377", "date": "15 Nov 2017", "name": "Richard Z. Lin", "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: The authors made a very interesting observation that L-DKO mice, lacking hepatic p110α and p85α, have a milder metabolic phenotype than mice lacking only hepatic p110α. These mice have normal fasting blood glucose levels but not surprisingly they are glucose intolerant. Interestingly, the liver and WAT weights were reduced. Liver triglyceride levels were not measured to determine if the fat content of the liver was also reduced. Metabolic cage measurements would be revealing to rule in or out changes in energy expenditure of the animals. It would also be informative to know if a high-fat diet causes L-DKO animals to develop fasting hyperglycemia and/or if the fatty liver associated with this type of diet is accentuated or attenuated.\n\nIn general, the results from the insulin signaling experiments are not convincing. It is unclear why the authors concluded that the residual signaling to Akt is not due to Class I PI3Ks. IRS-1 pull-downs from L-DKO liver have substantial PI kinase activity. Better experiments are needed to resolve this research question (see comment below).\n\nSpecific Comments: Figures 1F & 1G, Figure 2 and Figure 3E. (a) The dose of insulin used to study insulin signaling was extremely high. 5 U injected into a 30 g mouse is 167 U/kg body weight. Compare that to the 1.25 U/kg body weight used for the insulin tolerance test. Some of the signaling changes could be due to activation of an unusually high percentage of insulin receptors or insulin activation of IGF1 receptors that would not occur under physiological conditions. Attempts to explain the pathophysiological phenotypes of the L-DKO mouse based on the signaling results is therefore problematic. (b) The legends say that “Basal conditions refer to fasting of mice overnight.” Were the control mice anesthetized and injected ivc with saline, as stated under Methods? If so, the legends could be more specific.\n\nFigure 1F. Total Akt and total p70S6K should be shown.\n\nFigure 1G. (a) Why did insulin treatment of control mice not increase the amount of p85α (or other regulatory subunit) associated with IRS-1? (b) Use of a pan p85 antibody to estimate changes in p85β expression is not convincing. Lack of a good mouse p85β antibody is a limitation to the study. (c) The conclusion that similar amounts of IRS-1–bound p110β were pulled down in control and L-DKO mice is not convincing based on the blot.\n\nFigure 2. The conclusion that p110β and the other PI3Ks examined by western blotting are not responsible for the IRS-1–associated kinase activity is premature. The IPs should be assayed in the presence and absence of selective inhibitors to determine the contribution of p110β and the other enzymes to the PI kinase activity. Considering that hepatocytes make up 75-80% of the cell types in the liver, it is also possible that some of the kinase activity in the IRS-1 IP comes from p110α in the 20-25% of cells that are not targeted by albumin-Cre.\n\nFigure 3. Histological examination of the livers should be included. Liver triglyceride levels should be measured.\n\nFigures 3C & 3D. The specific WAT depots and muscles examined should be identified.\n\nFigure 4A-D. Fasting insulin is higher in the L-DKO mice at 10 weeks of age. Presumably, this is why these mice are euglycemic. Insulin levels should be measured before and after the glucose challenge with mice in one of the GTT age groups. This experiment will assess if the glucose intolerance is due to inability of the L-DKO mice to mount a further insulin response.\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": "3664", "date": "05 Jun 2018", "name": "Victoria Rotter Sopasakis", "role": "Author Response", "response": "Response to general comments: We agree with the reviewers that all these studies would be very interesting, but would take several years to complete and is outside the scope of this particular study. Response to specific comments: 1a. The dose of insulin used is what is normally used for these types of experiments. If the reviewers would be right, i.e unusually high activation of insulin would occur, this would be the case in all our other similar studies (and all other scientists’ studies who are doing similar experiments), for example in the study where we knocked out p110a in the liver. Since this is not the case, we don’t believe that this a major problem for the interpretation of our data. 1b. Yes, the control mice were anesthetized and injected ivc with saline (basal conditions) or insulin. This has been added to the figure legends as required. 2. Total Akt and total p70S6K blots have been added to Figure 1F. 3a. We have added more experiments to this figure (n=4) and made quantitative graphs of the results. The amount of p85a associated with IRS1 still remains similar between basal and insulin-stimulated conditions, whereas the p85-pan antibody shows increased amounts of total p85 in response to insulin (figures 1N and 1O). 3b. We have previously (before submitting the manuscript) tried various different p85b antibodies without any success. Some work well for human tumor tissue for example, but give no band at all for our mouse liver lysates. Other antibodies we tried fail to deliver a band for both human and mouse tissues. This is the reason we chose to use the p85-pan antibody to indirectly show p85b protein expression. Since both Dr Woodgett and Drs Lin and Ballou requested a p85b antibody we have tried again to make various p85b antibodies on the market to work for our liver lysates and have spent months now trying to optimize the western blot conditions for these antibodies. We are not satisfied with the results. The only conclusion we can draw is that either 1) the p85b antibodies available on the market simply does not work well for mouse tissue or 2) there is not much p85b expressed in mouse liver. Based on the p85-pan westerns that we performed as well as previous studies including Taniguchi et al, we believe that there simply is not that much p85b expressed in mouse liver. 3c. More experiments have been added (n=4) and quantifying graphs are now included in the figure (figure 1M). 4. These types of experiments are all very interesting. However, they are complicated, particularly considering that inhibitors are never 100% selective and specific. We therefore question the gain of these experiments in relation to the work and resources that have to be invested to complete them. 5. Histological experiments and liver triglyceride level measurements are outside the scope of this study. 6. These types of experiments would not lead to increased understanding of the underlying mechanisms of our data. We therefore question the gain of these experiments in relation to the work and resources that have to be invested to complete them. 7. It is quite possible that the L-DKO mice are not able to further increase the insulin secretion is response to the glucose load in the GTT considering the very high insulin levels these mice have normally. We have added a glucagon ELISA (new figure 4H) that shows normal glucagon levels for DKO mice both at 10 w of age and 24 w of age. The normal glucagon levels and the very high insulin levels in the DKO mice indicates that the pancreas is working normally in these mice and the underlying mechanism for the glucose intolerance is therefore not likely at the level of the pancreas." } ] } ]	1	https://f1000research.com/articles/6-1600
https://f1000research.com/articles/7-698/v1	04 Jun 18	{ "type": "Research Article", "title": "What does it mean if a patient is positive for anti-Jo-1 in routine hospital practice? A retrospective nested case-control study", "authors": [ "Paresh Jobanputra", "Feryal Malick", "Emma Derrett-Smith", "Tim Plant", "Alex Richter", "Feryal Malick", "Emma Derrett-Smith", "Tim Plant", "Alex Richter" ], "abstract": "Background: It is widely believed that patients bearing auto-antibodies to histidyl tRNA synthetase (anti-Jo-1) very likely have a connective tissue disease including myositis and interstitial lung disease. The value of positive tests in low disease prevalence settings such as those tested in routine care is unknown. We sought to determine the value of anti-Jo-1 auto-antibodies in routine practice. Methods: Our study was a nested case control study within a retrospective cohort of all patients tested for anti-ENA our hospital, from any hospital department, between January 2013 and December 2014. Data was extracted from electronic records of anti-Jo-1 positive patients and randomly selected ENA negative patients (ratio of 1:2), allowing for a minimum follow up of at least 12 months after first testing. Results: 4009 samples (3581 patients) were tested. Anti-ENA was positive in 616 (17.2%) patients, 40 (1.1%) were anti-Jo-1 positive. Repeat ENA testing was done for 350/3581 (9.8%) patients (428 of 4009 (10.7%) samples) and in 7/40 (17.5%) of anti-Jo-1 positive patients. The median interval between the first and second request was 124 days (inter-quartile range 233 days). The frequencies of interstitial lung disease (ILD), myositis and Raynaud’s were comparable for anti-Jo-1 positive patients (n=40) and 80 randomly selected ENA negative controls. Positive tests led to additional diagnostic testing in the absence of clinical disease. Sensitivity of Jo-1 for ILD was 50% (CI 19-81%), specificity 68% (CI 59-77%), positive predictive value 12.5% (CI 4 to 27%) and negative predictive value 93.8% (CI 86-98%). Of 10 (25%) patients with high anti-Jo1 levels, 3 had ILD, one myositis and two a malignancy (disseminated melanoma and CML). Conclusion: Anti-Jo-1 is uncommon in a heterogenous hospital population and is only weakly predictive for ILD. Repeated test requests were common and potentially unnecessary indicating that controls over repeat requests could yield significant cost savings.", "keywords": [ "Jo-1", "anti-histidyl-tRNA synthetase", "synthetase syndrome", "interstitial lung disease", "myositis", "connective tissue disease", "over diagnosis" ], "content": "Introduction\n\nDiagnosis of a connective tissue disease (CTD) may be considered in many clinical situations because of the diverse clinical manifestations of these diseases. Diagnosing a specific condition may be facilitated by a variety of immunological tests, including auto-antibodies to anti-nuclear antibodies (ANA) or extractable nuclear antigens (ENA)1,2. Anti-ENA antibodies consist of a panel including auto-antibodies associated with systemic lupus erythematosus, scleroderma, mixed connective tissue disease, myositis and Sjogren’s syndrome.\n\nData from the manufacturers of anti-ENA testing kits indicate that the diagnostic precision of anti-Jo-1 (sensitivity and specificity) exceeds 95%3. The sensitivity and specificity of a test does not tell us about the probability of disease. The sensitivity and specificity data of many diagnostic tests are calculated from case-control studies, in which test positivity is compared in patients with a known connective tissue disease with healthy controls or controls with other diseases. These methods greatly over-estimate the positive predictive value (PPV) of a diagnostic test, because disease prevalence is increased in the assembled population by the methods used for calculation4. Clinicians find diagnostic test accuracy data confusing and tend to over-estimate the probability of disease following a positive test result5. Disease prevalence in populations tested for anti-ENA in routine care is rarely known because testing may be requested and performed for any patient and without constraints in most hospitals. Thus, the prevalence of relevant diseases is likely to be considerably lower. Positive test results in general hospital practice may thus lead to over-diagnosis, especially in sick hospital patients with multiple morbidities, and may lead to additional expensive testing, patient and clinician anxiety and overtreatment.\n\nIn this study we focus on anti-Jo-1 (histidyl-tRNA synthetase). This autoantibody is associated with inflammatory muscle diseases and interstitial lung diseases (ILD). We retrospectively identified a cohort of patients tested for the panel of anti-ENA autoantibodies in our hospital. Using a nested case-control design we identified patients who tested positive for anti-Jo-1 and compared them with controls, from the same source population, who tested negative for anti-Jo-1. We sought to determine the accuracy and value of this test for hospital medical practice.\n\n\nMethods\n\nThe patient population (P) included in the present study was those in whom an ENA test was requested (I), from any department in Queen Elizabeth Hospital Birmingham (Birmingham, UK) over 2 years, between January 2013 and December 2014. The patient population was identified from records in the Immunology laboratory. This hospital is a large regional teaching hospital and referral center. Patients who were positive for anti-Jo-1 were the population of interest and randomly selected patients negative for anti-Jo-1 served as controls (C). The outcome of interest was a clinical diagnosis of myositis or ILD (O), determined by a clinical diagnosis of these conditions within patient medical records.\n\nENA tests are conducted sequentially by the University of Birmingham Immunology Laboratory. First, samples are screened for ENA auto-antibodies using the Quanta Lite® ENA 6 ELISA (Inova Diagnostics). Screen-negative patients are not tested further. Screen-positive patients have levels of auto-antibodies to ENA sub-types quantified by Quanta Lite products for each of the 6 ENAs screened: anti-Sjögren’s Syndrome A antigen (SSA); anti-Sjögren’s Syndrome B antigen (SSB); anti-Scl-70 antigen (also known as DNA-topoisomerase-1); anti-ribonucleoprotein (nRNP; also known as U1RNP), a small nuclear ribonucleoprotein; anti-Smith (Sm) antigen, another small nuclear ribonucleoprotein; and anti-Jo 1 (histidyl-tRNA synthetase). These assays are semi-quantitative, thus numerical values are available for each of the ENA subtypes if a sample proves to be ENA-screen-positive and are then tested in detail. For anti-Jo-1, levels >20 AU/ml are deemed to be positive, according to manufacturer instructions.\n\nStored serum samples were available for some anti-Jo-1-positive patients. We retrospectively re-tested these samples on a second occasion with the same Quanta Lite® platform to confirm anti-Jo-1 positivity and compared levels with the original test result. The first sample tested, during the study period, was designated as the test of interest.\n\nIf patients were tested for ENA on more than one occasion between January 2013 and December 2014, the numbers of repeat tests per patient were counted and the interval between the first and subsequent tests during this 2-year study period calculated. We compared levels of anti-Jo-1 for patients who were tested on a second occasion to assess the stability of anti-Jo-1 values. This was only possible in samples that were ENA-screen-positive on both occasions, since screen negative samples are not tested with semi-quantitative measurements, as per our laboratory protocol.\n\nClinical and radiographic data from positive patients was extracted from hospital electronic records. We ensured that a minimum of 12-month clinical follow up was available after the first ENA test. This allowed time for any planned clinical and diagnostic evaluations to be completed and allowed time for the evolution of the clinical picture. A random sample of ENA-screen-negative patients served as controls, in a ratio of 2 controls for each ENA positive patient. Control patients were selected using the random number generator in Microsoft Excel.\n\nOur study was registered with our hospital governance department (registration number CARMS-12140). Ethical approval was judged not necessary based on definitions provided by the NHS Health Research Authority6.\n\nCases (anti-Jo-1-positive patients) were identified from the population of anti-ENA-tested patients and controls (anti-Jo-1-negative patients) selected randomly from that same population. Thus, our study was a nested case-control study7. Supplementary File 1 contains a completed STARD checklist; Supplementary File 2 contains a completed STROBE checklist. We decided from the outset to include two controls for each case. Manufacturer data for sensitivity and specificity for the Quanta Lite product indicate that their anti-Jo-1 test is more than 99% specific and around 14% sensitive for polymyositis, yielding a positive likelihood ratio of 14. We assumed that these data hold true for our population and that each positive patient had disease (myositis and/or ILD) and that around 5% of controls had disease. Based on these assumptions and by substituting the likelihood ratio for the odds ratio, we estimated that around 18 cases and 54 controls would be needed to provide a power of 90% and alpha risk of 5%8.\n\nStatistical calculations were performed using Microsoft Excel 365. Descriptive statistics were used to compare key characteristics between patients and controls. Differences in proportions were compared using Fisher’s exact test. Where repeated testing was done for the same ENA positive patient, the result from the test of interest (first test during study period) was compared with the next available ENA test. This was done to evaluate test stability, using a Bland–Altmann plot. The diagnostic utility of anti-Jo-1 for a diagnosis of ILD and/or myositis, based on clinical diagnosis in patient records, was assessed by calculating sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV).\n\n\nResults\n\nA total of 4014 samples from 3584 patients were tested for ENA during the 2-year study period. There were 3 patients excluded because their samples originated from outside our hospital, leaving 4009 samples and 3581 patients. The first sample tested chronologically was designated the test of interest. Positive ENA tests occurred in 616 (17.2%) patients, 2965 (82.8%) patients were negative. The study flow diagram is shown in Figure 1. The frequency of positives at levels of ≥20 and ≥40 units are shown in Table 1. SSA, SSB, combined SSA and SSB, including patients with high concentrations of auto-antibodies, were commonly found in this population.\n\nOther combinations of ENA serotypes occurred in fewer than 10 patients for each of the combinations not shown here.\n\n1Arranged by frequency.\n\nWe studied the anti-Jo-1 positive patients (40, 1.1%) in detail and selected 80 ENA negative patients, randomly, as controls. Key clinical characteristics of these two populations are shown in Table 2.\n\nFisher’s exact test, two tailed. *Statistical analyses were not done for these comparisons, controls were negative for ENA antibodies, by definition.\n\nA total of 350 patients (9.8%; 428 samples or 10.7% of all samples) were tested on more than one occasion, 55 patients were tested on 3 or more occasions and 10 patients were tested on 4 or more occasions. Of the 40 (1.1%) anti-Jo-1-positive samples, 7 had the test done on more than one occasion. The median interval between the first and second test request for 350 patients was 124 days (interquartile range, 233 days; mean 168 days).\n\nThe stability of Jo-1 values between the first and second test, where a second test was requested later and where numerical data (ENA screen positive) were available, are shown in Figure 2. This illustrates that values remain stable between repeat tests over a median of 124 days, but that stability appears to lessen as anti-Jo-1 values rise. The likelihood of tests being repeated was greater in ENA-positive patients (18.8%) than in ENA-negative patients (7.8%), p<0.0001, Fisher’s exact test.\n\nJo-1 is considered positive if >20 AU/ml. The bold horizontal line shows the bias and dotted lines show upper and lower limits of agreement. Narrow limits of agreement are shown, but note that agreement appears to deteriorate at higher values of Jo-1. The bias or mean difference value (bold horizontal line) is small.\n\nFinally, using available stored samples from Jo-1-positive patients (n=33), we checked whether repeat measurement on the same sample using the same Quanta Lite® platform yielded consistent value (Figure 3). In three cases the first test result was reported as >50 U/ml (per laboratory practice at that time), whereas on retesting, more precise values were reported. For analysis, we assumed, in these strongly positive cases, that the original test results were identical to the more precise values reported on repeat testing. The degree of agreement between these repeat measures on the same sample is visualized using a Bland–Altman plot (Figure 3), the bias (average difference between repeat measures) was 5 AU/ml (95% CI, −8.0–18.7). We were concerned that auto-antibody levels in stored samples would have degraded over time; however, results from stored samples were not consistently lower or higher than the index test result (Figure 3).\n\nThe bold horizontal line shows the bias and dotted lines show upper and lower limits of agreement.\n\nClinical and demographic data all from 40 anti-Jo-1-positive patients was compared with 80 controls (ENA negative) (Table 2). The mean follow up time from the first anti-ENA test to review of medical records was 2.5 years (range, 1.6–3.5 years). When comparing the frequency of ILD, myositis and Raynaud’s, no statistically significant differences were found. The sensitivity and specificity of Jo-1 for ILD, as the key feature of ‘anti-synthetase syndrome’, were 50% (95% CI, 19–81%) and 68% (95% CI, 59–77%), respectively; the PPV was 12.5% (95% CI, 4–27%) and NPV was 93.8% (95% CI, 86–98%). Very few patients had other described features of anti-synthetase syndrome or the full spectrum of this disorder to calculate diagnostic accuracy data. There are no gold standards or widely accepted classification criteria for anti-synthetase syndrome9. Patients with anti-Jo-1 had a very wide range of co-morbidities (Table 3).\n\nIn a sub-group of patients with the highest anti-Jo-1 titers (≥40 AU/ml; 10/40 patients), 9 had a chest CT scan at some stage of their illness: one patient with metastatic melanoma and lung involvement including evidence of ILD and raised muscle enzymes was given a diagnosis of anti-synthetase syndrome; two other patients had ILD and normal muscle enzymes, one of these patients had systemic lupus erythematosus and lupus nephritis; one patient had chronic myeloid leukemia, one congestive cardiac failure and polymyalgia rheumatica, one Sjögren’s syndrome, two osteoarthritis (one with emphysema), and two with hepatitis (one labelled as autoimmune). Records indicated that two patients had chest CT scans primarily because of a positive anti-Jo-1, not because respiratory disease was found. In both these cases chest scans were normal. Only one of the anti-Jo-1-positive patients had a recorded creatine phosphokinase (CPK) level >1000 units/liter (the patient with metastatic melanoma)) and another had a diagnosis of myositis with a normal CPK level. Muscle biopsies were not done in any of these patients.\n\n\nDiscussion\n\nStudies such as this one, which report on the diagnostic value of a test in routine care and where patients have a wide variety of clinical features, are uncommon. Often, academic estimations of the diagnostic value of a test are assessed by comparing positivity in patients with confirmed disease against those with no disease or another disease. This approach increases the prevalence of diseased subjects in the population used to make calculations of diagnostic accuracy. Diagnostic sensitivity and specificity of a test may vary with disease prevalence and with disease spectrum, for example disease severity and the presence of co-morbidity4,11. Thus, a test may perform better where selected cohorts of patients with known disease and more severe disease are included, as seems likely to occur in cohorts of patients from referral centers, where much of the data on the relationship between Jo-1 and disease originate. The true value of a test only becomes apparent when it is used in usual clinical settings where diagnosis may be uncertain, other serious illnesses present, symptoms are unexplained or where there is multi-morbidity.\n\nThe PPV of anti-Jo-1 for ILD in our study was 12.5%. This figure is well below that produced by another study, in which anti-Jo-1 was concluded to be highly specific for autoimmune myositis but not thought to be sensitive12. Our patients were tested using a commercial ELISA for anti-ENA and anti-Jo-1. These assays have been compared with other methods and other manufacturers’ immunoassays, and are believed to be reliable13. Nevertheless, anti-Jo-1 detected by ELISA may lack specificity13,14. Older techniques are believed to be more specific and use native antigens in soluble form, whereas ELISAs and other assays, such as addressable laser bead immunoassays, use antigen-coated surfaces9. The use of a solid phase in ELISA could cause conformational changes to antigens or antigen denaturation, influencing test performance.\n\nA plea for greater clinical correlation and consideration before introducing new assays, particularly in the context of local test usage, was made over a decade ago15. Indeed, the UK National External Quality Assessment Service (UKNEQAS) recommends that a less sensitive or more specific test, such as indirect immunofluorescence (IIF), should be used to screen for samples prior to testing by more sensitive methods15. However, UKNEQAS reports considerable differences in the interpretation of IIF. Regarding ENA testing, UKNEQAS note: “a more worrying observation is the continuing complete lack of homogeneity and agreement of reported ENA specificities across the various manufacturers for almost any ENA type”16.\n\nAnti-Jo-1 is grouped with autoantibodies believed to be specific for myositis17, and occurs very rarely in normal populations18. Around 22% of patients with polymyositis diagnosed in specialist centers had anti-Jo-119 In addition when clinical data was reviewed for anti-Jo-1 positive patients, identified from a laboratory database, 70% had ILD20. This compares with 12.5% ILD in our anti-Jo-1-positive cohort. An association of myositis and ILD has led to coining of the term ‘anti-synthetase syndrome’. The notion of a unique syndrome attributable to the presence of auto-antibodies to synthetases has been challenged by a review of cohort studies9; our data add to this doubt. We showed that 17% of our population screened positive for anti-ENAs, most commonly SSA and or SSB. Anecdotally, we know that these positive tests result in referral from primary care to secondary care. Anti-Jo-1 occurred in 1.1% of our patients, including many without a recognized CTD, myositis or ILD.\n\nOur study has notable limitations. First, its retrospective design means that systematic data collection, for example specialist review and specific steps to determine the presence of Raynaud’s, ‘mechanics hands’, myositis or ILD, were not planned from the outset. Second, patients found to be positive for anti-Jo-1 could have been investigated more diligently than those found to be negative: an example of work up or verification bias. Thus, there could have been a greater propensity to diagnose ILD and use diagnostic tests such as chest CT scans. We identified two patients with high titer anti-Jo-1 who had such scans motivated purely by a positive anti-Jo-1 test.\n\nWe relied on routinely collected clinical data recorded in hospital electronic records and depended on clinical diagnoses rather than applying disease classification criteria. We did not access primary care records and may also have missed data if patients had been seen at other hospitals or in private health clinics. However, a strength of our study was the inclusion of a large cohort of patients seen in a variety of clinical settings. A retrospective design allowed our study to be completed with limited resources and in a timely manner. The inclusion of a heterogeneous population provides more reliable estimates of the value of anti-ENA and anti-Jo-1 in hospitals, the setting where these tests are likely to be ordered. We judged that a follow-up period of 1 year after the first anti-ENA test was done, provided sufficient time for key clinical features to emerge, especially ILD. We recognize, though, that auto-antibodies may precede CTD by many years and acknowledge that more prolonged follow-up may have given added value.\n\nWe show that anti-Jo-1 occurs in patients with varied illnesses, including cancer and other autoimmune diseases, raising the possibility that seriously ill patients with multiple morbidities may develop auto-antibodies at significant levels without relevant auto-immune diseases, but perhaps because of tissue damage. Auto-antibodies found in myositis have been classified into those believed to be specific for myositis (for example anti-Jo-1) and those that are associated with myositis. This distinction implies a special role for certain autoantibodies in disease pathogenesis. For anti-Jo-1, this view may be justified since this autoantibody can cause lung and muscle disease in animal models21. Yet, our data suggest that caution is necessary in according anti-Jo-1 a prominent role in disease pathogenesis. Recent research indicates that histidyl-tRNA synthetases have a role beyond protein synthesis, such as maintenance of immune homeostasis and stimulating immunological responses22,23. This suggests that much more needs to be learnt about the potential immunopathogenic role of anti-Jo-1 and other anti-synthetases.\n\nThe development, evaluation and dissemination of diagnostic tests is not standardized. Additionally, methods of test evaluation are often sub-optimal24. Test usage may become widespread and clinicians may not fully appreciate the limitations of a test in practice. A test believed to be highly specific for a disease owing to published or manufacturer data may lead to additional confirmatory diagnostic testing, including invasive testing, with the risk of over-diagnosis and patient harm.\n\nOf the anti-synthetase auto-antibodies, only anti-Jo-1 is tested routinely. A plea for wider serological testing, especially in patients with ILD, was made recently25. It is believed that patients with ILD who have auto-antibodies other than anti-Jo-1 have a worse prognosis. However, we believe that robust evaluations of the precision of newer auto-antibodies for diagnosis and determining prognosis, in heterogeneous populations likely to be tested in secondary care, are necessary before wider dissemination.\n\nWe found that 10.7% (428) of the samples tested were repeat requests for anti-ENA, in some cases on multiple occasions. The ‘Choosing Wisely’ initiative, supported by the American College for Rheumatology, recommends restraint and careful patient selection when first testing for ANA. Testing for anti-ENA is not recommended when the ANA is negative26. It appears, therefore, that widespread inappropriate laboratory testing is occurring. Determining the appropriateness of testing, however, is problematic27, but examples of good practice, at least for restricting repeated test requests, have been described by the UK Academy of Medical Colleges28. We did not attempt to ascertain reasons for repeated requests, but such testing may be done where there is diagnostic uncertainty, borderline results or simply because of inadequate review of available data or not knowing that samples had recently been submitted. For example, we show that 17.3% of anti-ENA repeat requests were made within 7 days of the first request.\n\nRestricting test requests or ‘gating’ has been implemented successfully for anti-neutrophil cytoplasm antibody (ANCA) in an English hospital29, though such a policy requires clinician and laboratory staff time, which may mean that such a strategy is not cost effective. However, a policy to decline repeat requests within specified time periods could be implemented more readily and could be automated. Our hospital is in the process of implementing this for selected immuno-diagnostic tests.\n\nLimited data suggests that anti-Jo-1 levels reflect disease activity30. Our data shows that anti-Jo-1 levels measured by ELISA vary at higher concentrations when re-checked at a later point (Figure 2). We also noted variation in test results when the same positive sample was re-tested (Figure 3) after storage for many months.\n\nIn summary, we show that many hospital patients negative for ANA are tested for anti-ENA, often repeatedly. We show that positivity for anti-Jo-1 is uncommon and that positive results are poorly predictive for interstitial lung disease and clinically diagnosed autoimmune myositis.\n\n\nData availability\n\nDataset 1. Complete data for all participants regarding disease status and results of extractable nuclear antigen (ENA) testing. Data are presented as ENA-negative controls, Jo-1-positive patients and data pooled from all patients. DOI: 10.5256/f1000research.14834.d20425410.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nNo grants were involved in the funding of this work.\n\n\nAcknowledgements\n\nPreliminary data from this study were published by the British Society for Rheumatology in abstract form in 2016 (https://doi.org/10.1093/rheumatology/kew186.001) and were presented at the European League Against Rheumatism in 2017 (https://dx.doi.org/10.1136/annrheumdis-2017-eular.2939).\n\n\nSupplementary material\n\nSupplementary File 1. Completed STARD checklist.\n\nClick here to access the data.\n\nSupplementary File 2. Completed STROBE checklist.\n\nClick here to access the data.\n\n\nReferences\n\nAmerican College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines: Guidelines for immunologic laboratory testing in the rheumatic diseases: an introduction. Arthritis Rheum. 2002; 47(4): 429–33. 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Mod Rheumatol. 2008; 18(2): 153–160. PubMed Abstract \| Publisher Full Text\n\nChinoy H, Salway F, Fertig N, et al.: In adult onset myositis, the presence of interstitial lung disease and myositis specific/associated antibodies are governed by HLA class II haplotype, rather than by myositis subtype. Arthritis Res Ther. 2006; 8(1): R13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDugar M, Cox S, Limaye V, et al.: Clinical heterogeneity and prognostic features of South Australian patients with anti-synthetase autoantibodies. Intern Med J. 2011; 41(9): 674–9. PubMed Abstract \| Publisher Full Text\n\nKatusmata Y, Ridgway WM, Oriss T, et al.: Species-specific immune responses generated by histidyl-tRNA synthetase immunization are associated with muscle and lung inflammation. J Autoimmunity. 2007; 29(2–3): 174–186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBecker R: Beyond building proteins: tRNA synthetases outside of translation. Nat Med. 2016; 22(5): 452–3. PubMed Abstract \| Publisher Full Text\n\nZhou JJ, Wang F, Xu Z, et al.: Secreted histidyl-tRNA synthetase splice variants elaborate major epitopes for autoantibodies in inflammatory myositis. J Biol Chem. 2014; 289(28): 19269–275. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReid MC, Lachs MS, Feinstein AR: Use of methodological standards in diagnostic test research. Getting better but still not good. JAMA. 1995; 274(8): 645–51. PubMed Abstract \| Publisher Full Text\n\nCotton CV, Spencer LG, New RP, et al.: The utility of comprehensive autoantibody testing to differentiate connective tissue disease associated and idiopathic interstitial lung disease subgroup cases. Rheumatology (Oxford). 2017; 56(8): 1264–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolomon DH, Kavanaugh AJ, Schur PH, et al.: Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum. 2002; 47(4): 434–444. PubMed Abstract \| Publisher Full Text\n\nZhi M, Ding EL, Theisen-Toupal J, et al.: The landscape of inappropriate laboratory testing: A 15-year meta-analysis. PLoS One. 2013; 8(11): e78962. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAcademy of Medical Royal Colleges: Protecting resources, promoting value: a doctor’s guide to cutting waste in clinical care. 2014; [cited 2017 Dec 17]. Reference Source\n\nArnold DF, Timms A, Luqmani R, et al.: Does a gating policy for ANCA overlook patients with ANCA associated vasculitis? An audit of 263 patients. J Clin Path. 2010; 63(8): 678–80. PubMed Abstract \| Publisher Full Text\n\nHervier B, Benveniste O: Clinical heterogeneity and outcomes of antisynthetase syndrome. Curr Rheumatol Rep. 2013; 15(8): 349. PubMed Abstract \| Publisher Full Text" }	[ { "id": "34691", "date": "13 Jun 2018", "name": "Robert Cooper", "expertise": [ "Reviewer Expertise Correlations of serology with myositis and ILD phenotypes" ], "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 constructed piece of research which examines the diagnostic value (sensitivity and selectivity) of anti-Jo-1 positivity when detected by standard ENA testing in a large teaching hospital. The results clearly show that Jo-1 positivity is not particularly predictive of myositis or ILD, as many other non-CTD conditions can also be associated with a positive result. This is rather surprising, but the authors explain why this outcome. The reviewer has no negative comments to make about this article, but is not a statistical expert, so the view of a statistician may be warranted.\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": "34692", "date": "23 Jul 2018", "name": "Stephen Adelstein", "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 address an important question about the performance characteristics and clinical utility of autoantibody testing. They review the outcome of ENA testing requested in a general hospital where the tested patients apparently do not have a high probability for the disease associated with a specific ENA of interest, Jo-1. The paper is well-written and highlights many of the problems in the use and interpretation of autoantibody tests, particularly the use of an auto-antibody test to define a disease subgroup.\nTheir findings are not surprising, concluding that there is no difference in the clinical associations with those patients in the cohort who were ENA negative compared to those who were ENA positive. It is well known that tests do not perform well when inappropriately selected in patients who do not have at least a reasonable pre-test probability of the disease in question. This paper illustrates this very well, but does not emphasize this aspect sufficiently in the discussion of the results.\nOne other issue not discussed by the authors that likely impacts on their findings is the performance in laboratories of ENA tests as a group. In the authors laboratory a request for anti-ENA antibodies produces a request for a screen of antibodies to six ENAs, and if this test is positive, further tests are performed to identify to which of the six ENAs the antibodies bind. This is not an uncommon strategy in Immunology laboratories, but ignores the pre-test probability of diseases that prompted request for ENA testing. It is likely that many of the requests for ENA received in the laboratory were for diagnosis of other autoimmune diseases rather than myositis and ILD and that has clinicians had a choice of which ENA antibody to request they may not have chosen anti-Jo-1 antibodies as the clinical pre-test probability for requesting this ENA was low. The test was, nevertheless, performed because of the testing algorithm in operation in the laboratory. This scenario would no doubt affect the calculated positive predictive value in the paper.\nIt would, therefore, be misleading from the information provided in this study, to conclude that anti-Jo 1 antibodies do not perform well when requested in the appropriate circumstances, that is, in individuals who have evidence of myositis and/or interstitial lung disease. In these patients a positive result for Jo-1 antibodies may be helpful in selecting appropriate treatment and in prognostication.\nThis study highlights that anti-Jo-1 positivity, in the absence of clear clinical features, has a very low predictive value. As mentioned by the authors, a longer follow up period may be useful to determine whether there is any delayed predictive value of anti-Jo-1, like findings reported with anti-CCP positivity and rheumatoid arthritis.\nAlthough a much larger study would be required, it would have been interesting to correlate the authors’ findings with the rate of anti-Jo-1 positivity in patients already diagnosed with interstitial lung disease, at the same hospital.\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/7-698
https://f1000research.com/articles/6-1618/v1	31 Aug 17	{ "type": "Opinion Article", "title": "Best practice data life cycle approaches for the life sciences", "authors": [ "Philippa C. Griffin", "Jyoti Khadake", "Kate S. LeMay", "Suzanna E. Lewis", "Sandra Orchard", "Andrew Pask", "Bernard Pope", "Ute Roessner", "Keith Russell", "Torsten Seemann", "Andrew Treloar", "Sonika Tyagi", "Jeffrey H. Christiansen", "Saravanan Dayalan", "Simon Gladman", "Sandra B. Hangartner", "Helen L. Hayden", "William W.H. Ho", "Gabriel Keeble-Gagnère", "Pasi K. Korhonen", "Peter Neish", "Priscilla R. Prestes", "Mark F. Richardson", "Nathan S. Watson-Haigh", "Kelly L. Wyres", "Neil D. Young", "Maria Victoria Schneider", "Jyoti Khadake", "Kate S. LeMay", "Suzanna E. Lewis", "Sandra Orchard", "Andrew Pask", "Bernard Pope", "Ute Roessner", "Keith Russell", "Torsten Seemann", "Andrew Treloar", "Sonika Tyagi", "Jeffrey H. Christiansen", "Saravanan Dayalan", "Simon Gladman", "Sandra B. Hangartner", "Helen L. Hayden", "William W.H. Ho", "Gabriel Keeble-Gagnère", "Pasi K. Korhonen", "Peter Neish", "Priscilla R. Prestes", "Mark F. Richardson", "Nathan S. Watson-Haigh", "Kelly L. Wyres", "Neil D. Young" ], "abstract": "Throughout history, the life sciences have been revolutionised by technological advances; in our era this is manifested by advances in instrumentation for data generation, and consequently researchers now routinely handle large amounts of heterogeneous data in digital formats. The simultaneous transitions towards biology as a data science and towards a ‘life cycle’ view of research data pose new challenges. Researchers face a bewildering landscape of data management requirements, recommendations and regulations, without necessarily being able to access data management training or possessing a clear understanding of practical approaches that can assist in data management in their particular research domain.\nHere we provide an overview of best practice data life cycle approaches for researchers in the life sciences/bioinformatics space with a particular focus on ‘omics’ datasets and computer-based data processing and analysis. We discuss the different stages of the data life cycle and provide practical suggestions for useful tools and resources to improve data management practices.", "keywords": [ "data sharing", "data management", "open science", "bioinformatics", "reproducibility" ], "content": "Introduction\n\nTechnological data production capacity is revolutionising biology1, but is not necessarily correlated with the ability to efficiently analyse and integrate data, or with enabling long-term data sharing and reuse. There are selfish as well as altruistic benefits to making research data reusable2: it allows one to find and reuse one’s own previously-generated data easily; it is associated with higher citation rates3,4; and it ensures eligibility for funding from and publication in venues that mandate data sharing, an increasingly common requirement (e.g. Final NIH statement on sharing research data,Wellcome Trust policy on data management and sharing,Bill & Melinda Gates Foundation open access policy). Currently we are losing data at a rapid rate, with up to 80% unavailable after 20 years5. This affects reproducibility - assessing the robustness of scientific conclusions by ensuring experiments and findings can be reproduced - which underpins the scientific method. Once access to the underlying data is lost, replicability, reproducibility and extensibility6 are reduced.\n\nAt a broader societal level, the full value of research data may go beyond the initial use case in unforeseen ways7,8, so ensuring data quality and reusability is crucial to realising its potential value9–12. The recent publication of the FAIR principles9,13 identifies four key criteria for high-quality research data: the data should be Findable, Accessible, Interoperable and Reusable. Whereas a traditional view of data focuses on collecting, processing, analysing data and publishing results only, a life cycle view reveals the additional importance of finding, storing and sharing data11. Throughout this article, we present a researcher-focused data life cycle framework that has commonalities with other published frameworks [e.g. the DataONE Data Life Cycle, the US geological survey science data lifecycle model and 11,14–15], but is aimed at life science researchers specifically (Figure 1).\n\nBlack arrows indicate the ‘traditional’, linear view of research data; the green arrows show the steps necessary for data reusability. This framework is likely to be a simplified representation of any given research project, and in practice there would be numerous ‘feedback loops’ and revisiting of previous stages. In addition, the publishing stage can occur at several points in the data life cycle.\n\nLearning how to find, store and share research data is not typically an explicit part of undergraduate or postgraduate training in the biological sciences16–18. The scope, size and complexity of datasets in many fields has increased dramatically over the last 10–20 years, but the knowledge of how to manage this data is currently limited to specific cohorts of ‘information managers’ (e.g. research data managers, research librarians, database curators and IT professionals with expertise in databases and data schemas18). In response to institutional and funding requirements around data availability, a number of tools and educational programs have been developed to help researchers create Data Management Plans to address elements of the data lifecycle19; however, even when a plan is mandated, there is often a gap between the plan and the actions of the researcher10.\n\nDuring the week of 24–28 October 2016, EMBL Australia Bioinformatics Resource (EMBL-ABR)20 led workshops on the data life cycle for life science researchers working in the plant, animal, microbial and medical domains. The workshops provided opportunities to (i) map the current approaches to the data life cycle in biology and bioinformatics, and (ii) present and discuss best practice approaches and standards for key international projects with Australian life scientists and bioinformaticians. Discussions during these workshops have informed this publication, which targets life science researchers wanting to improve their data management practice; throughout we highlight some specific data management challenges mentioned by participants.\n\nAn earlier version of this article can be found on bioRxiv (https://doi.org/10.1101/167619).\n\n\nFinding data\n\nIn biology, research data is frequently published as supplementary material to articles, on personal or institutional websites, or in non-discipline-specific repositories like Figshare and Dryad21. In such cases, data may exist behind a paywall, there is no guarantee it will remain extant, and, unless one already knows it exists and its exact location, it may remain undiscovered22. It is only when a dataset is added to public data repositories, along with accompanying standardized descriptive metadata (see Collecting data), that it can be indexed and made publicly available23. Data repositories also provide unique identifiers that increase findability by enabling persistent linking from other locations and permanent association between data and its metadata.\n\nIn the field of molecular biology, a number of bioinformatics-relevant organisations host public data repositories. National and international-level organisations of this kind include the European Bioinformatics Institute (EMBL-EBI)24, the National Centre for Biotechnology Information (NCBI)25, the DNA Data Bank of Japan (DDBJ)26, the Swiss Institute of Bioinformatics (SIB)27, and the four data center members of the worldwide Protein Data Bank28, which mirror their shared data with regular, frequent updates. This shared central infrastructure is hugely valuable to research and development. For example, EMBL-EBI resources have been valued at over £270 million per year and contribute to ~£1 billion in research efficiencies; a 20-fold return on investment29.\n\nNumerous repositories are available for biological data (see Table 1 for an overview), though repositories are still lacking for some data types and sub-domains30. Many specialised data repositories exist outside of the shared central infrastructure mentioned, often run voluntarily or with minimal funding. Support for biocuration, hosting and maintenance of these smaller-scale but key resources is a pressing problem31–33. The quality of the user-submitted data in public repositories34,35 can mean that public datasets require extra curation before reuse. Unfortunately, due to low uptake of established methods (see the EMBL-EBI and NCBI third-party annotation policies and;36) to correct the data35, the results of extra curation may not find their way back into the repositories. Repositories are often not easily searched by generic web search engines30. Registries, which form a secondary layer linking multiple, primary repositories, may offer a more convenient way to search across multiple repositories for data relevant to a researcher’s topics of interest37.\n\n\nCollecting data\n\nThe most useful data has associated information about its creation, its content and its context - called metadata. If metadata is well structured, uses consistent element names and contains element values with specific descriptions from agreed-upon vocabularies, it enables machine readability, aggregation, integration and tracking across datasets: allowing for Findability, Interoperability and Reusability9,30. One key approach in best-practice metadata collection is to use controlled vocabularies built from ontology terms. Biological ontologies are tools that provide machine-interpretable representations of some aspect of biological reality30,38. They are a way of organising and defining objects (i.e. physical entities or processes), and the relationships between them. Sourcing metadata element values from ontologies ensures that the terms used in metadata are consistent and clearly defined. There are several user-friendly tools available to assist researchers in accessing, using and contributing to ontologies (Table 2).\n\nAdopting standard data and metadata formats and syntax is critical for compliance with FAIR principles9,23,30,37,39. Biological and biomedical research has been considered an especially challenging research field in this regard, as datatypes are extremely heterogeneous and not all have defined data standards39,40; many existing data standards are complex and therefore difficult to use40, or only informally defined, and therefore subject to variation, misrepresentation, and divergence over time39. Nevertheless, well-established standards exist for a variety of biological data types (Table 3). FAIRsharing is a useful registry of data standards and policies that also indicates the current status of standards for different data types and those recommended by databases and research organisations37.\n\nMost public repositories for biological data (see Table 1 and Storing data section) require that minimum metadata be submitted accompanying each dataset (Table 4). This minimum metadata specification typically has broad community input43. Minimum metadata standards may not include the crucial metadata fields that give the full context of the particular research project43, so it is important to gather metadata early, understand how to extend a minimum metadata template to include additional fields in a structured way, and think carefully about all the relevant pieces of metadata information that might be required for reuse.\n\n\nProcessing and analysing data\n\nRecording and reporting how research data is processed and analysed computationally is crucial for reproducibility and assessment of research quality1,44. Full reproducibility requires access to the software, software versions, dependencies and operating system used as well as the data and software code itself45. Therefore, although computational work is often seen as enabling reproducibility in the short term, in the long term it is fragile and reproducibility is limited (e.g. discussion by D. Katz, K. Hinsen and C.T. Brown). Best-practice approaches for preserving data processing and analysis code involve hosting source code in a repository where it receives a unique identifier and is under version control; where it is open, accessible, interoperable and reusable - broadly mapping to the FAIR principles for data. Github and Bitbucket, for example, fulfil these criteria, and Zenodo additionally generates Digital Object Identifiers (DOIs) for submissions and guarantees long-term archiving. Several recent publications have suggested ways to improve current practice in research software development15,46–48.\n\nThe same points hold for wet-lab data production: for full reproducibility, it is important to capture and enable access to specimen cell lines, tissue samples and/or DNA as well as reagents. Wet-lab methods can be captured in electronic laboratory notebooks and reported in the Biosamples database49, protocols.io or OpenWetWare; specimens can be lodged in biobanks, culture or museum collections50–54; but the effort involved in enabling full reproducibility remains extensive. Electronic laboratory notebooks are frequently suggested as a sensible way to make this information openly available and archived55. Some partial solutions exist (e.g. LabTrove, BlogMyData, Benchling and others56), including tools for specific domains such as the Scratchpad Virtual Research Environment for natural history research57. Other tools can act as or be combined to produce notebooks for small standalone code-based projects [Boettiger, 201758 and update], including Jupyter Notebook, Rmarkdown, and Docker. However, it remains a challenge to implement online laboratory notebooks to cover both field/lab work and computer-based work, especially when computer work is extensive, involved and non-modular44. Currently, no best-practice guidelines or minimum information standards exist for use of electronic laboratory notebooks6. We suggest that appropriate minimum information to be recorded for most computer-based tasks should include date, task name and brief description, aim, actual command(s) used, software names and versions used, input/output file names and locations, script names and locations.\n\nDuring the EMBL-ABR workshop series, participants identified the data processing and analysis stage as one of the most challenging for openness. A few participants had put intensive individual effort into developing custom online lab (and code) notebook approaches, but the majority had little awareness of this as a useful goal. This suggests a gap between modern biological research as a field of data science, and biology as it is still mostly taught in undergraduate courses, with little or no focus on computational analysis, or project or data management. As reported elsewhere16–18, this gap has left researchers lacking key knowledge and skills required to implement best practices in dealing with the life cycle of their data.\n\n\nPublishing data\n\nTraditionally, scientific publications included raw research data, but in recent times datasets have grown beyond the scope of practical inclusion in a manuscript11,44. Selected data outputs are often included without sharing or publishing the underlying raw data14. Journals increasingly recommend or require deposition of raw data in a public repository [e.g. 59], although exceptions have been made for publications containing commercially-relevant data60. The current data-sharing mandate is somewhat field-dependent5,61 and also varies within fields62. For example, in the field of bioinformatics, the UPSIDE principle63 is referred to by some journals (e.g. Bioinformatics), while others have journal- or publisher-specific policies (e.g. BMC Bioinformatics).\n\nThe vast majority of scientific journals require inclusion of processing and analysis methods in ‘sufficient detail for reproduction’ (e.g. Public Library of Science submission and data availability guidelines; International Committee of Medical Journal Editors manuscript preparation guidelines; Science instructions for authors; Elsevier Cell Press STAR Methods; and64), though journal requirements are diverse and complex65, and the level of detail authors provide can vary greatly in practice66,67. More recently, many authors have highlighted that full reproducibility requires sharing data and resources at all stages of the scientific process, from raw data (including biological samples) to full methods and analysis workflows1,6,53,67. However, this remains a challenge68,69, as discussed in the Processing and analysing data section. To our knowledge, strategies for enabling computational reproducibility are currently not mandated by any scientific journal.\n\nA recent development in the field of scientific publishing is the establishment of ‘data journals’: scientific journals that publish papers describing datasets. This gives authors a vehicle to accrue citations (still a dominant metric of academic impact) for data production alone, which can often be labour-intensive and expensive yet is typically not well recognised under the traditional publishing model. Examples of this article type include the Data Descriptor in Scientific Data and the Data Note in GigaScience, which do not include detailed new analysis but rather focus on describing and enabling reuse of datasets.\n\nThe movement towards sharing research publications themselves (‘Open Access Publishing’) has been discussed extensively elsewhere [e.g. 22,70,71]. Publications have associated metadata (creator, date, title etc.; see Dublin Core Metadata Initiative metadata terms) and unique identifiers (PubMed ID for biomedical and some life science journals, DOIs for the vast majority of journals; see Table 5). The ORCID system enables researchers to claim their own unique identifier, which can be linked to their publications. The use of unique identifiers within publications referring to repository records (e.g. genes, proteins, chemical entities) is not generally mandated by journals, although it would ensure a common vocabulary is used and so make scientific results more interoperable and reusable72. Some efforts are underway to make this easier for researchers: for example, Genetics and other Genetics Society of America journals assist authors in linking gene names to model organism database entries.\n\n\nStoring data\n\nWhile primary data archives are the best location for raw data and some downstream data outputs (Table 1), researchers also need local data storage solutions during the processing and analysis stages. Data storage requirements vary among research domains, with major challenges often evident for groups working on taxa with large genomes (e.g. crop plants), which require large storage resources, or on human data, where privacy regulations may require local data storage, access controls and conversion to non-identifiable data if data is to be shared (see the Australian National Data Service de-identification guide, the National Health and Medical Research Council statement on ethical conduct in human research, and the Australian National Medical Research Storage Facility discussion paper on legal, best practice and security frameworks). In addition, long-term preservation of research data should consider threats such as storage failure, mistaken erasure, bit rot, outdated media, outdated formats, loss of context and organisational failure73.\n\n\nSharing data\n\nThe best-practice approach to sharing biological data is to deposit it (with associated metadata) in a primary archive suitable for that datatype8 that complies with FAIR principles. As highlighted in the Storing data section, these archives assure both data storage and public sharing as their core mission, making them the most reliable location for long-term data storage. Alternative data sharing venues (e.g. FigShare, Dryad) do not require or implement specific metadata or data standards. This means that while these venues have a low barrier to entry for submitters, the data is not FAIR unless submitters have independently decided to comply with more stringent criteria. If available, an institutional repository may be a good option if there is no suitable archive for that datatype. Importantly, plans for data sharing should be made at the start of a research project and reviewed during the project, to ensure ethical approval is in place and that the resources and metadata needed for effective sharing are available at earlier stages of the data life cycle3.\n\nDuring the EMBL-ABR workshop series, the majority of participants were familiar with at least some public primary data repositories, and many had submitted data to them previously. A common complaint was around usability of current data submission tools and a lack of transparency around metadata requirements and the rationale for them. A few workshop participants raised specific issues about the potential limitations of public data repositories where their data departed from the assumptions of the repository (e.g. unusual gene models supported by experimental evidence that were rejected by the automated NCBI curation system). Most workshop participants were unaware they could provide feedback to the repositories to deal with such situations, and this could also be made clearer on the repository websites. Again, this points in part to existing limitations in the undergraduate and postgraduate training received by researchers, where the concepts presented in this article are presented as afterthoughts, if at all. On the repository side, while there is a lot of useful information and training material available to guide researchers through the submission process (e.g. the EMBL-EBI Train Online webinars and online training modules), it is not always linked clearly from the database portals or submission pages themselves. Similarly, while there are specifications and standards available for many kinds of metadata [Table 4; also see FAIRsharing], many do not have example templates available, which would assist researchers in implementing the standards in practice.\n\n\nWhat can the research community do to encourage best-practice?\n\nWe believe that the biological/biomedical community and individual researchers have a responsibility to the public to help advance knowledge by making research data FAIR for reuse9, especially if the data were generated using public funding. There are several steps that can assist in this mission:\n\n1. Senior scientists should lead by example and ensure all the data generated by their laboratories is well-managed, fully annotated with the appropriate metadata and made publicly available in an appropriate repository.\n\n2. The importance of data management and benefits of data reuse should be taught at the undergraduate and postgraduate levels18. Computational biology and bioinformatics courses in particular should include material about data repositories, data and metadata standards, data discovery and access strategies. Material should be domain-specific enough for students to attain learning outcomes directly relevant to their research field.\n\n3. Funding bodies are already taking a lead role in this area by requiring the incorporation of a data management plan into grant applications. A next step would be for a formal check, at the end of the grant period, that this plan has been adhered to and data is available in an appropriate format for reuse10.\n\n4. Funding bodies and research institutions should judge quality dataset generation as a valued metric when evaluating grant or promotion applications.\n\n5. Similarly, leadership and participation in community efforts in data and metadata standards, and open software and workflow development should be recognised as academic outputs.\n\n6. Data repositories should ensure that the data deposition and third-party annotation processes are as FAIR and painless as possible to the naive researcher, without the need for extensive bioinformatics support35.\n\n7. Journals should require editors and reviewers to check manuscripts to ensure that all data, including research software code and samples where appropriate, have been made publicly available in an appropriate repository, and that methods have been described in enough detail to allow re-use and meaningful reanalysis8.\n\n8. Finally, researchers reusing any data should openly acknowledge this fact and fully cite the dataset, including unique identifiers8,10,30.\n\n\nConclusions\n\nWhile the concept of a life cycle for research data is appealing from an Open Science perspective, challenges remain for life science researchers to put this into practice. During the EMBL-ABR Data Life Cycle workshop series, we noted limited awareness among attendees of the resources available to researchers that assist in finding, collecting, processing, analysis, publishing, storing and sharing FAIR data. We believe this article provides a useful overview of the relevant concepts and an introduction to key organisations, resources and guidelines to help researchers improve their data management practices.\n\nFurthermore, we note that data management in the era of biology as a data science is a complex and evolving topic and both best practices and challenges are highly domain-specific, even within the life sciences. This factor may not always be appreciated at the organisational level, but has major practical implications for the quality and interoperability of shared life science data. Finally, domain-specific education and training in data management would be of great value to the life science research workforce, and we note an existing gap at the undergraduate, postgraduate and short course level in this area.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis publication was possible thanks to funding support from the University of Melbourne and Bioplatforms Australia (BPA) via an Australian Government NCRIS investment (to EMBL-ABR).\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 thank Dan Bolser for his involvement in the EMBL-ABR Data Life Cycle workshops, and all workshop participants for sharing their experiences and useful discussions.\n\n\nReferences\n\nCohen-Boulakia S, Belhajjame K, Collin O, et al.: Scientific workflows for computational reproducibility in the life sciences: status, challenges and opportunities. 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Reference Source" }	[ { "id": "27111", "date": "21 Nov 2017", "name": "Johannes Starlinger", "expertise": [ "Reviewer Expertise Biomedical knowledge management", "systems architectures", "clinical informatics" ], "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 gives a brief overview of the data life cycle in the life sciences and offers an entry point for accessing relevant information about current approaches to increasing compliance with the FAIR data sharing principles at each step of this life cycle. It expressly targets \"life science researchers wanting to improve their data management practice\" and is labeled as an Opinion article.\n\nThe article is well written and comfortable to read, and the concise presentation follows a clear structure. While to me as a biomedical data researcher, who may not strictly belong to the target audience, the article provided only little additional insight, I can well see how - as an entry point - the article provides valuable information to its target audience.\nThat said, I believe the article needs clarification and some extension in a few places:\n\nThe list of authors is quite extensive. Please clarify the roles of the authors in conception/conduction/preparation of the manuscript.\n\nHow exactly does the proposed data life cycle differ from related (cited) suggestions, and why? How is it 'aimed at life science researchers specifically'? (Introduction)\n\nThe tabular overviews of existing resources are a nice asset but they are, of course, not exhaustive. Please clarify how the selections of databases/registries, tools, ontologies etc were made for inclusion in the article - and possibly state where to find more complete lists of resources for the life sciences.\n\nThe integrating step of the life cycle has no description in the article - even though this is a very intricate step that often has great influence when collecting data (e.g., the choice of ontologies to use for describing collected data and metadata will often depend on the ontologies used in re-used (found) data), and, even more, is at the core of making datasets interoperable, i.e., making them integratable with newly collected data.\n\nIn the processing step, you make no mention of Scientific Workflows as a means of integrating, processing, and analyzing data. Your first reference (currently cited in a rather different context) would provide a very good hook for this thriving topic that is all about sharing, reproducibility, and reusability of data processing and analysis methods. On the same lines, containerized computing (e.g., Docker) is only very briefly metioned. Even more than with data, using technologies such as these is crucial for ensuring reproducibility over longer periods of time (when software versions of dependencies have changed, web-services have become unavailable, and so forth).\n\nThe section \"What can the research community do to encourage best practice?\" gives a rather remote, high level view that addresses several different institutional entities - except for the individual researcher within the target audience who actually has to follow the discussed best practices to enable the data life cycle.\n\nAdditionally, here are some suggestions for increasing the usefulness and potential impact of the article within the current target audience, and possibly beyond:\nImportant interdependecies between the different steps of the life cycle could be mentioned. For instance, the choice of which ontologies to use for metadata and data in the collection step will necessarily be influenced by a) the ontologies used in the data found in public repositiories and reused in the current experiment, b) the ontologies mandated by the repositories the data product is to be published in, and c) the ontologies required and used by the (third party, reused) software applied in the processing of the data. These interdependencies often not only put a limit to the choices available regarding the ontologies to be used but also raise a barrier when conversion and mapping between different ontologies is necessary between steps in the life cycle.\n\nThe topic of data privacy is only very briefly touched but fundamental when it comes to sharing and publishing data. It may be out of scope of this article, but a slightly more thorough discussion of the issue would to its importance more justice, I feel.\n\nAn additional figure that maps the best practices enumerated in the text to the rather coarse life cycle shown in Figure 1 could prove highly instructive. Something like a 'data life cycle best practices cheat sheet' ;)\n\nIf you (the authors) have any questions regarding this review, please do not hesitate to contact me.\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": "3683", "date": "04 Jun 2018", "name": "Pip Griffin", "role": "Reader Comment", "response": "Response to Review 2 We thank Dr. Starlinger for his review and respond to his comments below (reviewer comments in italics, our responses in plain text). The article gives a brief overview of the data life cycle in the life sciences and offers an entry point for accessing relevant information about current approaches to increasing compliance with the FAIR data sharing principles at each step of this life cycle. It expressly targets \"life science researchers wanting to improve their data management practice\" and is labeled as an Opinion article. The article is well written and comfortable to read, and the concise presentation follows a clear structure. While to me as a biomedical data researcher, who may not strictly belong to the target audience, the article provided only little additional insight, I can well see how - as an entry point - the article provides valuable information to its target audience. Thank you. That said, I believe the article needs clarification and some extension in a few places: The list of authors is quite extensive. Please clarify the roles of the authors in conception/conduction/preparation of the manuscript. The authorship roles are described in the ‘Author Details’ section using the F1000Research authorship classification scheme. To give a bit more detail, Maria Victoria Schneider and Philippa Griffin conceptualised the paper as a follow-up to the Data Life Cycle workshop series run by EMBL Australia Bioinformatics Resource (EMBL-ABR) in October 2016. Jyoti Khadake, Suzanna Lewis, Sandra Orchard, Andrew Pask, Bernard Pope, Ute Roessner, and Torsten Seemann were workshop faculty who presented sessions and led group discussions. Jeffrey Christiansen, Sonika Tyagi, Nathan Watson-Haigh, Saravanan Dayalan and Simon Gladman have Key Area Coordinator roles with EMBL-ABR. All other authors were workshop attendees who subsequently volunteered to contribute to the manuscript. Philippa Griffin drafted the manuscript with input and supervision from Maria Victoria Schneider. All authors then had the opportunity to edit and comment on the text, figures and tables (via a shared Google Doc) and did so through several revisions of the manuscript. How exactly does the proposed data life cycle differ from related (cited) suggestions, and why? How is it 'aimed at life science researchers specifically'? (Introduction) This data life cycle is rather similar to others but we see it as having some important practical differences that make it more relevant to life science researchers, as follows: The USGS data life cycle model does not include distinct steps for finding existing data and collecting new data (both are implied under ‘acquire’), whereas in the life sciences these two steps are performed differently, with different limitations and considerations and so we see the need for highlighting both. As Dr. Nahnsen (the other reviewer) has noted, the integration of existing and new data can also be very complex in the life sciences and so deserves a place in the data life cycle diagram (it does not occur in the USGS model). Finally, we have separated the ‘publish’ and ‘share’ steps since publishing manuscripts and sharing data are highly distinct in the minds of most life science researchers due to the publication-focussed way the world of life science research currently operates. Each has different actions relevant to good practice data management. The DataOne data life cycle model has a heavier focus on data collection, with distinct steps for ‘collect’, ‘assure’, and ‘describe’. We would argue that data quality assurance is generally considered an intrinsic part of data collection in the life sciences and does not require its own step. We also consider ‘description’ as part of the collection step as this should be done at the same time (or ideally planned beforehand), and we cover this in the article with the sections on metadata. This model also lacks the Publishing and Sharing steps (‘sharing’ is subsumed with ‘storing’ under ‘preserve’) which we believe are important, distinct considerations for life science researchers as mentioned above. The Digital Curation Centre data lifecycle model is similar to the DataOne model. The tabular overviews of existing resources are a nice asset but they are, of course, not exhaustive. Please clarify how the selections of databases/registries, tools, ontologies etc were made for inclusion in the article - and possibly state where to find more complete lists of resources for the life sciences. These tables are intended to demonstrate the scope of the resources available and indeed are not exhaustive. The databases/registries, standards and ontologies presented were ‘crowd-sourced’ from the authors’ suggestions, in an attempt to present the most relevant options for resources used across the wide range of biology sub-domains this group of authors represents. We have now referenced FAIRsharing.org in the caption of the databases/registries and standards tables (Tables 1, 3 and 4), as this website contains more complete, maintained lists of resources. The integrating step of the life cycle has no description in the article - even though this is a very intricate step that often has great influence when collecting data (e.g., the choice of ontologies to use for describing collected data and metadata will often depend on the ontologies used in re-used (found) data), and, even more, is at the core of making datasets interoperable, i.e., making them integratable with newly collected data. We have now changed the title of the Processing and Analysing Data section to Integrating, processing and analysing data to ensure this step is highlighted. The point about integration having great influence on the data collection and processing strategy is indeed important and we have now included a paragraph dealing with this explicitly (Integrating, Processing and Analysing Data section, para 1). In the processing step, you make no mention of Scientific Workflows as a means of integrating, processing, and analyzing data. Your first reference (currently cited in a rather different context) would provide a very good hook for this thriving topic that is all about sharing, reproducibility, and reusability of data processing and analysis methods. On the same lines, containerized computing (e.g., Docker) is only very briefly metioned. Even more than with data, using technologies such as these is crucial for ensuring reproducibility over longer periods of time (when software versions of dependencies have changed, web-services have become unavailable, and so forth). We agree this is an active and important area of development in the research reproducibility field. We have now expanded the Integrating, Processing and Analysing Data section (para 2) to include mention of scientific workflows, workflow repositories and containerized computing. The section \"What can the research community do to encourage best practice?\" gives a rather remote, high level view that addresses several different institutional entities - except for the individual researcher within the target audience who actually has to follow the discussed best practices to enable the data life cycle. Thanks for pointing this out - we have now added three recommendations for individual researchers at the start of this section as follows: 1. Researchers reusing any data should openly acknowledge this fact and fully cite the dataset, including unique identifiers.2. Researchers should endeavour to improve their own data management practices in line with best practice in their subdomain - even incremental improvement is better than none!3. Researchers should provide feedback to their local institution, data repositories and bodies responsible for community resources (data formats, controlled vocabularies etc.) where they identify roadblocks to good data management. Additionally, here are some suggestions for increasing the usefulness and potential impact of the article within the current target audience, and possibly beyond: Important interdependecies between the different steps of the life cycle could be mentioned. For instance, the choice of which ontologies to use for metadata and data in the collection step will necessarily be influenced by a) the ontologies used in the data found in public repositiories and reused in the current experiment, b) the ontologies mandated by the repositories the data product is to be published in, and c) the ontologies required and used by the (third party, reused) software applied in the processing of the data. These interdependencies often not only put a limit to the choices available regarding the ontologies to be used but also raise a barrier when conversion and mapping between different ontologies is necessary between steps in the life cycle. At the risk of making the paper too long, we agree it is important to point out the complexities and interdependencies that can be involved in good data management practice (this actually helps explain why it is implemented rather haphazardly at present). We have now included a flow-chart (Figure 2) as a guide to how a researcher might actually use a data life cycle approach. It is still rather high-level but shows how downstream requirements influence choices made at each stage of a research project. The topic of data privacy is only very briefly touched but fundamental when it comes to sharing and publishing data. It may be out of scope of this article, but a slightly more thorough discussion of the issue would to its importance more justice, I feel. We agree that data privacy is fundamental for research involving human data and have now expanded the text in the Finding Data section (para 3), the Storing Data section (para 1) and the Sharing Data section (para 1) to make it clear to readers that for human data, much extra planning and effort is typically required to deal with these considerations. An additional figure that maps the best practices enumerated in the text to the rather coarse life cycle shown in Figure 1 could prove highly instructive. Something like a 'data life cycle best practices cheat sheet' ;) We are concerned a generic ‘cheat sheet’ would not incorporate enough subdomain-specific detail to be of practical use. Instead, we’ve included a ‘flow chart’ figure (now Figure 2) to demonstrate an example of how a researcher might work through the data life cycle - including feedback loops that show the need for prior planning. If you (the authors) have any questions regarding this review, please do not hesitate to contact me." } ] }, { "id": "27113", "date": "15 Dec 2017", "name": "Sven Nahnsen", "expertise": [ "Reviewer Expertise Data management", "multi-omics bioinformatics" ], "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 \"Best practice data life cycle approaches for the life sciences\", submitted by Griffin et al. reports opinions on how to best manage the growing complexity of scientific data in the life sciences.\nThe article touches on an extremely important topic that is currently very purely covered in the literature. In fact, data-driven approaches in the biosciences will strongly rely on professional concepts of data management. In brief, I recommend the indexing of the article, as we urgently need stronger awareness of this topic, upon the implementation of some (probably rather minor) changes to the article. The article nicely illustrates the needs in data life cycle management and also suggests best concepts to be followed by researchers. The main content of the article has been compiled based on a workshop that was attended by the authors. At some statements the article reads like the minutes of this meeting; I suggest editing the corresponding paragraphs to avoid the impression of reading meeting minutes.\nI suggest the following issues to be fixed before indexing:\nFigure 1: This illustration is very important and can be used by many readers. I suggest to use figures wherever possible to replace the words such as “finding”, “integrating”, …\n\nThe reference to Figure 1 in the second paragraph states that it illustrates a specific aim to the life sciences. I don’t see which of these points should be specific to the life science, but would rather argue that these principles are rather generic and provides a cycle for business intelligence processes in general. It might also be a good location to reference the DAMA (Data management association internation, dama.org) and specifically to the DAMA Body of Knowledge, which is one of the few references for data management and also data life cycle considerations. Further needed references should hint to the Global Alliance for Genomics and Health (ga4gh.org).\n\nPage 13: The paragraph on data sharing missing some discussion on authentication issues. I would like see some introduction and discussion to the OpenID concept. Especially for medical data there need to be appropriate mechanisms to trace users, concepts for data privacy and so on. As a best practice use case for these topics, the mechanism from ICGC could be introduced.\n\nThe following paragraph states: “A few workshop participants…”. Rephrase, no meeting minutes..\n\nI would have loved to see more use cases/examples for the individual best practices. E.g. for the data sharing the ICGC efforts could be described more thoroughly.\n\nThe article would benefit for 2-3 additional figures. I guess it could be a nice figure to illustrate the concept of controlled vocabularies and/or ontologies. While this seems to be trivial for bioinformaticians/computer scientists, it is not that obvious what it means to non-computer scientists; inspiration for figures can also be obtained by the data sharing mechanisms for the Global alliance for Genomics and Health\n\nMinor things:\nThe forth paragraph in the introduction starts with “During the week of 24-28 October 2016…”. I suggest either avoiding that paragraph or formulating it differently. The reader should not be reading the meeting minutes.\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": "3682", "date": "04 Jun 2018", "name": "Pip Griffin", "role": "Reader Comment", "response": "Response to Review 1 Thank you very much to Dr. Nahnsen for his review. We have responded to his comments below (reviewer comments in italics, our responses in plain text). The article \"Best practice data life cycle approaches for the life sciences\", submitted by Griffin et al. reports opinions on how to best manage the growing complexity of scientific data in the life sciences. The article touches on an extremely important topic that is currently very purely covered in the literature. In fact, data-driven approaches in the biosciences will strongly rely on professional concepts of data management. In brief, I recommend the indexing of the article, as we urgently need stronger awareness of this topic, upon the implementation of some (probably rather minor) changes to the article. The article nicely illustrates the needs in data life cycle management and also suggests best concepts to be followed by researchers. Thank you. The main content of the article has been compiled based on a workshop that was attended by the authors. At some statements the article reads like the minutes of this meeting; I suggest editing the corresponding paragraphs to avoid the impression of reading meeting minutes. We have edited the Introduction (para 4), Integrating, Processing and Analysing Data section (para 4), the Sharing Data section (para 2) and the Conclusions section (para 1) to remove details of the events of the workshop, while still mentioning briefly in the Introduction (para 4) that this article arose from the material we presented and discussed in this workshop series. I suggest the following issues to be fixed before indexing: Figure 1: This illustration is very important and can be used by many readers. I suggest to use figures wherever possible to replace the words such as “finding”, “integrating”, … We experimented with adding icons to represent the life cycle stages, but found it too difficult to choose a single icon to summarise each complex stage. (For example: the ‘Storing Data’ stage text covers local data storage, primary archives, privacy and security considerations; one icon would necessarily omit or de-emphasise some of these.) Our attempts gave a misleading aura of simplicity, which we wanted to avoid, and so we prefer to retain the words in the figure, which map readily to the text of the article which contains the detail. The reference to Figure 1 in the second paragraph states that it illustrates a specific aim to the life sciences. I don’t see which of these points should be specific to the life science, but would rather argue that these principles are rather generic and provides a cycle for business intelligence processes in general. It might also be a good location to reference the DAMA (Data management association internation, dama.org) and specifically to the DAMA Body of Knowledge, which is one of the few references for data management and also data life cycle considerations. Further needed references should hint to the Global Alliance for Genomics and Health (ga4gh.org). We agree that data life cycle principles can cut across disciplines and have mentioned other examples of published data lifecycle figures in the Introduction, para 2. As described in more detail in our response to Dr. Starlinger’s review, we believe that our data lifecycle model is better suited to the way life science researchers work than more generic models. Specifically, we have included distinct steps for finding existing data and collecting new data (different from e.g. the USGS data lifecycle model) because in life science research these two steps typically have different limitations and considerations. We have included distinct ‘publish’ and ‘share’ steps (unlike the USGS, DataOne and Digital Curation Centre models) since publishing manuscripts and sharing data are highly distinct in the minds of most life science researchers due to the publication focus of life science research. Some models (e.g. the DataOne and Digital Curation Centre models) break down the ‘collecting data’ step (e.g. into collecting, quality-assuring and describing data) but we believe these stages are already rather well understood to be part of the data collection process in the life sciences and have kept them together. We have been unable to find GA4GH publications dealing with the research data lifecycle but have now cited GA4GH documents in the Storing Data (para 1) and Sharing Data (para 1) sections. We have been unable to source a copy of DAMA International’s Guide to the Data Management Body of Knowledge (https://technicspub.com/dmbok/) and so have not included this reference. Page 13: The paragraph on data sharing missing some discussion on authentication issues. I would like see some introduction and discussion to the OpenID concept. Especially for medical data there need to be appropriate mechanisms to trace users, concepts for data privacy and so on. As a best practice use case for these topics, the mechanism from ICGC could be introduced. In the interests of keeping the paper a concise introduction to the concepts, we decided not to delve into too much detail around data privacy considerations, a topic that indeed warrants entire papers to itself. However we have now expanded the text in the Finding Data section (para 3), the Storing Data section (para 1) and the Sharing Data section (para 1) to make it clear to readers that for medical data, much extra planning and effort is required to deal with these considerations. We have also provided some explanation of why authentication might be necessary, links to some of the relevant technologies, and a reference (as suggested) to the practices of the ICGC. The following paragraph states: “A few workshop participants…”. Rephrase, no meeting minutes.. Done (Sharing Data section, para 2). I would have loved to see more use cases/examples for the individual best practices. E.g. for the data sharing the ICGC efforts could be described more thoroughly. As the paper is aimed at individual researchers, we wanted to avoid an excessive focus on large-scale research consortium efforts, as the resources such projects have available for data management are likely to be far beyond what individual researchers can access. However, we acknowledge these efforts often set a ‘best-practice’ standard and so we have now mentioned the Monarch Initiative (Integrating, Processing and Analysing Data section, para 1), the GA4GH (Sharing Data section, para 1) and the ICGC (cited in Finding Data section, para 2). The article would benefit for 2-3 additional figures. I guess it could be a nice figure to illustrate the concept of controlled vocabularies and/or ontologies. While this seems to be trivial for bioinformaticians/computer scientists, it is not that obvious what it means to non-computer scientists; inspiration for figures can also be obtained by the data sharing mechanisms for the Global alliance for Genomics and Health We have now included a second figure, an example flowchart (Figure 2) showing how the data life cycle might be used in practice and how downstream considerations influence choices made at each step. An extra figure illustrating CVs/ontologies we judged would make the paper somewhat unbalanced - we have referenced other articles (Thessen and Paterson 2001, Malone et al. 2016) that are good starting points for researchers keen to learn about this topic. The forth paragraph in the introduction starts with “During the week of 24-28 October 2016…”. I suggest either avoiding that paragraph or formulating it differently. The reader should not be reading the meeting minutes. We have retained some reference to the origin of this article but rewritten the paragraph (Introduction, para 4) to avoid an appearance of meeting minutes." } ] } ]	1	https://f1000research.com/articles/6-1618
https://f1000research.com/articles/7-696/v1	04 Jun 18	{ "type": "Research Article", "title": "Autonomic nervous system assessment in people with HIV: A cross-sectional study", "authors": [ "Martin G. Rosario", "Maryvi Gonzalez-Sola", "Maryvi Gonzalez-Sola" ], "abstract": "Background: People diagnosed with HIV may exhibit orthostatic hypotension (OH) as a result of the infection and of secondary effects of medications. Such impairments are attributed to autonomic nervous system (ANS) deficits. The purpose of this study was to assess OH during a balance sensory condition test (SCT) and evaluate the role of the cardiac autonomic system, regarding blood pressure (BP) and heart rate (HR), during this balancing task. We hypothesized that BP and heart rate would rise with an increase in postural instability, thus revealing OH. Methods: Eight individuals diagnosed with HIV were recruited from a community health center in the area of San Juan, Puerto Rico. BP and HR were measured after 5 minutes of sitting, immediately after standing up and 1 minute after this, during the SCT. A t-test was used to assess the difference between BP in sitting, BP in standing, and BP while performing the SCT. HR was also evaluated the same way. Results: There was an increase of more than 10 mmHg in systolic BP (SBP) from sitting compared to standing while performing the SCT (p≤0.01). Likewise, HR and SBP standing versus standing during the SCT increased significantly (p≤0.01). Conclusion: The results of this study show that the ANS may be impaired in people with HIV.", "keywords": [ "HIV", "orthostatic hypotension", "ANS impairments", "blood pressure", "postural instability", "heart rate" ], "content": "Introduction\n\nHIV can destroy or substantially affect essential cells (CD4+ T cells or T helper cells) of the immune system, which ordinarily help combat diseases. The virus spreads through the exchange of certain bodily fluids such as blood or as a result of unprotected sex. HIV affects more than 34 million people in the world according to WHO statistics, and more than 3.3 million North Americans aged 13 years and older, according to CDC statistics (Centers for Disease Control and Prevention, 2015). There is no cure for this disease, and if left untreated, it can progress to AIDS (acquired immune deficiency syndrome, defined as 200 or fewer CD4+ cells/µl); however, medication to control HIV exists (antiretroviral therapy) which can prolong the lives of patients (Song et al., 2017; WHO, 2011).\n\nPatients with HIV can develop symptoms that vary depending on the disease stage. The most common symptoms are fever, fatigue, weight loss, swollen lymph nodes and shingles, amongst others. Typically, neurological complications, such as difficulty walking, are often seen in patients with advanced stages of HIV or AIDS. Nevertheless, there is one symptom that is not often mentioned, which it is known to affect HIV patients: orthostatic hypotension (OH) (Cohen et al., 1991; Robinson-Papp et al., 2013; Song et al., 2017; WHO, 2011).\n\nBy definition, OH is a reduction in blood pressure (BP) of at least 20 mmHg systolic or 10 mmHg diastolic after either 1 or 3 minutes of standing when the patient moves from supine or to the upright position. Symptoms of OH are often associated with medications, dehydration, and infections, as explained by Cohen et al., 1991. One theory of why OH may affect patients with HIV is due to impairments in the autonomic nervous system (ANS), which is typically responsible for the modulation of numerous functions, including heart rate and vasomotor tone (Robinson-Papp et al., 2013).\n\nAlthough neurologic complications due to HIV are well characterized in the central and peripheral nervous systems, it is not entirely understood in the ANS due to its complexity. However, studies show that autonomic dysfunctions (Cohen et al., 1991) and autonomic neuropathy (Robinson-Papp et al., 2013) are common in patients with HIV. The symptoms of autonomic neuropathy, therefore, could include orthostatic dizziness or fainting, among others (Robinson-Papp et al., 2013).\n\nAlthough there is some evidence of a connection between OH and impairments to the ANS, this connection is unclear and poorly understood. Therefore, the objective of this study is to evaluate OH during balance sensory condition tests (SCTs) and to assess the function of the cardiac autonomic system, such as BP and heart rate (HR), during a balance task. We hypothesized that BP and HR would rise with a change in postural positioning, thus revealing ANS dysfunction.\n\n\nMethods\n\nThe study protocol was approved by the Institutional Review Board of the University of Puerto Rico Medical Sciences Campus (Protocol A2540114). Authorization was given by the Executive Director of the Institution. Every participant read and signed an informed consent document after being informed of all risks and discomforts related to their participation in this investigation.\n\nWe recruited eight Puerto Rican participants with a diagnosis of HIV from a community health center, La Perla de Gran Precio, in the San Juan area of Puerto Rico. The Physical Therapist from the Center identified possible participants for this study during May–July 2016, who then telephoned M.G.R. to confirm recruitment into the study. Subjects were of both sexes (4 males and 4 females), with age (±SD) =48.7±9.7 years, CD4 count =599.6±225.5 cells/µl, time of diagnosis =15.6±6.2 years and BMI of 21.8±2.8.\n\nThe study inclusion criteria were: i) HIV diagnosed by a medical doctor; ii) male or female; iii) aged 21–65 years old; and iv) CD4+ T cell count equal to or higher than 200. The exclusion criteria were: i) CD4+ T cell count lower than 200; ii) inability to walk; iii) severe balance impairment (≥2 falls within the last 6 months); iv) lower limb amputations; v) injury or surgery of lower limbs or back in the last 6 months; vi) lower limb ulcer(s) in the last 6 months; vii) pregnant women or women trying to get pregnant (women were asked if they were pregnant or there could be a chance there could be at the moment of the test); viii) any surgery in the past year; and ix) any hospitalization within the last year.\n\nAfter providing written informed consent at the community-based fitness center in San Juan, Puerto Rico, anthropometric measurements were obtained from each participant. The anthropometric measures included height and weight which were measured with a scale and stadiometer. Following the anthropometric measures (height and weight), a sensibility assessment was performed with a monofilament on both feet.\n\nIn addition to the exclusion and inclusion criteria aforementioned, these too will serve as a more accurate criterion for the study. The screening assessments are described as follows:\n\nParticipants with a body mass index (BMI) of 18.5 or below (underweight) and 30.0 and above (obese) were excluded from the study. Thus, participants were required to have a BMI of 18.5–29.9.\n\nA Semmes-Weinstein monofilament was used to evaluate the sensation threshold on the foot area. The monofilament was applied perpendicular to the skin's surface. Neuropathy can be detected using the 5.07 monofilament (this filament bends with the application of a 10-g force). Participants unable to detect the 5.07 monofilament (loss of protective sensation and deep pressure sensation) in more than two areas were excluded from the study (Bell-Krotoski et al., 1993)\n\nAn iHealth Wireless Blood Pressure Monitor (iHealth Model: BP5) (a wireless BP monitor for the arm) was used to measure systolic BP (SBP) and diastolic BP (DBP), and heart rate.\n\nSBP, DBP and heart rate (HR) were measured after 5 minutes of sitting, immediately after standing up on the floor (firm surface), and 1 minute after beginning the SCT. In the SCT, patients were asked to close their eyes and actively nod their head up and down while standing on foam (an unstable surface), which eliminated visual and proprioceptive input and stimulated the vestibular system. The participants were instructed, while standing on foam, to sustain a set frequency (approximately one turn per second with a metronome, 60 bpm) and amplitude (about 45 degrees in each direction for flexion and extension) of movement to ensure the average velocity of movement was maintained. Once the participants got the rhythm, they were instructed to close their eyes for 30 seconds.\n\nThe comparisons in this study were as follows: First, we compared BP and HR during the sitting position and standing position. Secondly, we compared, BP and HR during the sitting position with standing position during the SCT. Lastly, we examined BP and HR in subjects while they were standing during the SCT.\n\nStatistical analysis was performed using SPSS version 20 software package for Windows. A paired sample Student’s t-test was used to assess the difference between BP sitting and BP standing, and standing versus standing during the SCT. HR was also assessed the same way. We considered a P-value of 0.01 as significant.\n\n\nResults\n\nWe analyzed eight subjects of different genders and years of HIV diagnosis from a health center in San Juan, Puerto Rico (Table 1). Raw data of heart rate, SBP and DBP is reported in Table 2. The average of BP and HR measurements for each subject before and after standing to observe possible ANS deficits associated with HIV (Table 2–Table 3).\n\nBMI, body mass index.\n\nSBP, systolic BP; DBP, diastolic BP.\n\nSBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate.\n\nWe assessed HR (Figure 1), and SBP (Figure 2) and DSB (Figure 3) to determine whether there were ANS alterations in these participants.\n\nST, sitting; SU, standing; SCT, standing while performing the sensory condition test.\n\nST, sitting; SU, standing; SCT, standing while performing the sensory condition test.\n\nST, sitting; SU, standing; SCT, standing while performing the sensory condition test.\n\nAll comparison are reported in Table 4. Results revealed no difference in BP and HR in sitting versus standing. However, there was a significant increase in SBP (mean ± SD) (SP, 136.5±17.8; BT, 153.7±16.5; P<0.01) and HR (SP, 73.9±10.1; BT, 109.7±13.9; P<0.05) when subjects changed positions from standing to standing during the SCT (see Figure 2). Additionally, in our last comparison, standing versus standing during the SCT, there was a significant increase in SBP (SP, 139.4±16.2; BT, 153.8±16.5; P<0.01) and HR (SP, 78.9±14.7; BT, 109.7±1.9; P<0.05).\n\nSBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate.\n\n\nDiscussion\n\nWe hypothesized that BP and heart rate would increase with changes in postural positioning, thus revealing ANS dysfunction. The key findings of our study were that participants demonstrated an increase in SBP and HR when going from sitting to standing while performing the SCT. There was also an increase in SBP and HR from standing to standing while carrying out the SCT. Therefore, our hypothesis was accepted. The present study provides support for the presence of autonomic dysfunction in persons with HIV.\n\nThe fact that SBP and HR changed from sitting and standing (on foam) could, by themselves, be causing alterations in the cardiovascular system. When we stand, muscles in charge of venous return in the lower extremities are activated (Recek, 2013). It is possible that muscle weakness is responsible for improper venous return, thus causing an increase in HR as a compensatory mechanism (Dymarek et al., 2014). However, the fact that BP and HR changed from standing to standing while performing the SCT was surprising. Head movements during the SCT could be affecting the carotid body (baroreceptor), thus resulting in fluctuation of BP and HR (Ogoh et al., 2003). However, it is important to point out that normal head tilts or movements do not cause BP changes (Ogoh et al., 2006) like the ones observed in the present study of subjects with HIV.\n\nVenous return can also be influenced by the difficulty experienced by individuals standing on a foam/complaint surface or their inability to do so. Lower extremities muscles will co-contract, thus increasing muscle activity to maintain the standing position. (Shumway-Cook & Woollacott, 2007).\n\nPrevious studies suggest that ANS alterations in patients with HIV are linked to the use of medications to control the HIV virus itself (Cohen et al., 1991). Other studies also show that there are neurologic deficits in people with HIV, specifically with the ANS. Although the ANS regulates many different bodily functions, we were most interested in identifying alterations within the ANS during balance tests and the cardiac autonomic response (BP and HR) during the same task. However, there is a lack of evidence concerning why OH affects many people with HIV.\n\nAn initial study by Cohen et al. in 1991 suggests that patients with HIV will have OH as a feature of ANS impairments. An update is needed to address the recent data related to OH and HIV. Therefore, we studied BP and HR in patients with HIV to reveal OH alteration. In our initial results, there was an increase in BP when participants were asked to go from a sitting to standing position, albeit not a significant one. It is possible that with these changes in BP and HR our participants with HIV are exhibiting ANS alterations or dysfunctions. Despite the different methodology used in this study, our results had similar findings compared to the information available in the literature (Cohen et al., 1991).\n\nIn this study, there was a significant increase (p<0.01) in HR when standing compared to HR when standing and performing the SCT. These results were surprising because such values have never been reported before. This study shows that the ANS may be impaired in people with HIV; however, more research is needed to confirm these findings.\n\nSome of the clinical implications of this study indicate the need for further investigation of the neuromuscular activation of lower extremity muscles responsible for antero-posterior sway (tibialis anterior/gastrocnemius) and venous return (gastrocnemius). Additionally, peripheral neuropathy testing should be considered when evaluating ANS changes in persons with HIV. Towards identifying ANS dysfunctions in this population, clinicians should observe changes in HR and BP during functional movements, such as moving from a supine position to sitting, and from sitting to standing.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "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 would like to thank ‘La Perla de Gran Precio' for their constant support and to the patients who were willing to participate in this study.\n\nThis article was published with support from Texas Woman's University Libraries’ Open Access Fund.\n\n\nReferences\n\nBell-krotoski J, Weinstein S, Weinstein C: Testing sensibility, including touch-pressure, two-point discrimination, point localization, and vibration. J Hand Ther. 1993; 6(2): 114–23. PubMed Abstract \| Publisher Full Text\n\nCenters for Disease Control and Prevention: Estimated HIV Incidence and Prevalence in the United States, 2010–2015. 2015; Retrieved on April 30, 2018. Reference Source\n\nCohen JA, Miller L, Polish L: Orthostatic hypotension in human immunodeficiency virus infection may be the result of generalized autonomic nervous system dysfunction. J Acquir Immune Defic Syndr. 1991; 4(1): 31–3. PubMed Abstract \| Publisher Full Text\n\nDymarek R, Ptaszkowski K, Słupska L, et al.: [Physiotherapy potentials improve the calf muscle pump function in chronic venous insufficiency]. Wiad Lek. 2014; 67(2 Pt 1): 112–8. PubMed Abstract\n\nOgoh S, Volianitis S, Nissen P, et al.: Carotid baroreflex responsiveness to head-up tilt-induced central hypovolaemia: effect of aerobic fitness. J Physiol. 2003; 551(Pt 2): 601–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOgoh S, Yoshiga CC, Secher NH, et al.: Carotid-cardiac baroreflex function does not influence blood pressure regulation during head-up tilt in humans. J Physiol Sci. 2006; 56(3): 227–33. PubMed Abstract \| Publisher Full Text\n\nRecek C: Calf pump activity influencing venous hemodynamics in the lower extremity. Int J Angiol. 2013; 22(1): 23–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson-Papp J, Sharma S, Simpson DM, et al.: Autonomic dysfunction is common in HIV and associated with distal symmetric polyneuropathy. J Neurovirol. 2013; 19(2): 172–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShumway-Cook A, Woollacott MH: Motor control: Translating research into clinical practice. Philadelphia: Lippincott Williams & Wilkins. 2007. Reference Source\n\nSong R, Hall HI, Green TA, et al.: Using CD4 Data to Estimate HIV Incidence, Prevalence, and Percent of Undiagnosed Infections in the United States. J Acquir Immune Defic Syndr. 2017; 74(1): 3–9. PubMed Abstract \| Publisher Full Text\n\nWHO: Global HIV/AIDS response: epidemic update and health sector progress towards universal access: progress report 2016. 2011; Retrieved April 24, 2018, 11. Reference Source" }	[ { "id": "39013", "date": "15 Oct 2018", "name": "Roger C. McIntosh", "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 report an increase in heart rate and SBP in HIV+ individuals for sitting compared to standing while performing the SCT. This fluctuation in cardioautonomic functioning is interpreted as orthostatic hypertension. The analyses seems appropriate however, the study has some limitations that appear to mitigate the impact of the paper.\nBesides the small sample size this study may be have been prone to selection bias. The authors report \"The Physical Therapist from the Center identified possible participants for this study\". The lack of randomization provided by a general recruiting strategy may have resulted in a bias to select HIV patients for the study based upon symptomology, thus oversampling for OH. This is a serious limitation and should be mentioned to the reader.\n\nWere the patients virally-suppressed? Were they on standard anti-retroviral therapy and for how long?\n\nWith regards to the cardio-metabolic function what percentage of the sample were hypertensive as this may perpetuate the blood pressure responses to postural adjustments.\n\nIt is perplexing why a control group was not recruited, particularly due to the small sample size of HIV+ individuals.\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? 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?\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/7-696
https://f1000research.com/articles/3-316/v1	29 Dec 14	{ "type": "Research Article", "title": "Qualia as social effects of minds", "authors": [ "Sheila Bouten", "J. Bruno Debruille", "Sheila Bouten" ], "abstract": "Qualia, the individual instances of subjective conscious experience, are private events. However, in everyday life, we assume qualia of others and their perceptual worlds, to be similar to ours. One way this similarity is possible is if qualia of others somehow contribute to the production of qualia by our own brain and vice versa. To test this hypothesis, we focused on the mean voltages of event-related brain potentials (ERPs) in the time-window of the P600 component, whose amplitude correlates positively with conscious awareness. These ERPs were elicited by stimuli of the international affective picture system in 16 pairs of friends, siblings or couples going side by side through hyperscanning without having to interact. Each member of each pair faced one half of the screen and could not see what the other member was presented with on the other half. One stimulus occurred on each half simultaneously. The sameness of these two stimuli was manipulated as well as the participants’ belief in that sameness. ERPs were more negative over left frontal sites and P600 amplitudes were minimal at midline sites when the two stimuli were, and were believed to be, different, suggesting that this belief could filter others’ qualia. ERPs were less negative over left frontal sites and midline P600s were a bit larger when the two stimuli were, and were believed to be, the same, suggesting some mutual enrichment of the content of awareness in conditions of real and assumed similarity. When stimuli were believed to be the same but actually differed, P600s were greater over a large number of sites, suggesting greater enrichment in conditions of qualia difference and assumed similarity. P600s were also larger over many sites, when stimuli were believed to differ but were identical, suggesting that qualia similar to ours could pass the “believed-different filter”.", "keywords": [ "Really direct brain-to-brain communications", "Mind-to-mind Qualia", "Consciousness", "Event-related brain potentials (ERPs)", "P3b-P600", "IAPS stimuli" ], "content": "Introduction\n\nColors, sounds and smells do not exist in the outside world. They are the creations of our brain in response to light waves, rhythmic variations of air pressure and inhaled molecules, respectively. External stimulations are responsible for action potentials whose processing in the brain may then produce colors, sounds and smells. These, so-called qualia1,2 are then apparently projected outside around us and constitute our perceptual world, sometimes called the phenomenal world3. Although perceived internally, except in the case of induced out-of-body experiences4, events, such as feelings of meanings, as in the tip of the tongue phenomenon, or such as emotions, conscious intentions to act and sensations of our body can also be seen as qualia.\n\nUnderstanding consciousness as consisting of self made qualia leads to one of the most enduring philosophical questions: Are qualia the same across individuals? In other words, is the yellow produced by the brain of one person the same as the yellow produced by the brain of another person? Surprisingly, there is no way to know for sure. The fact that the same word is used by all the people speaking a language to designate a qualia merely establishes a correspondence. It does not prevent the qualia it indicates from varying across these people. The yellow qualia for one person could, for instance, be the blue qualia for another person. Nevertheless, such differences across individuals appear unlikely since many use the same associations and agree that red is a warm color and that blue relates to sadness. In the auditory modality, many associate high pitch sounds with sharpness. Moreover, we use the same metaphor and define these sounds as “high” whereas those of longer wavelengths are said to be “low” sounds (for other metaphors see for instance Lakoff and Johnson5). The same relations between qualia thus seem to exist across people whereas if qualia were different across individuals it seems that these relations should differ. It could be agued that metaphors used in a language convey relationships between certain qualia and are thus responsible for building the links between them. However, it seems that new metaphors can be understood at their first occurrence6, which suggests that relations between qualia are, at least partly, independent of language.\n\nIn any case, if the qualia produced by our brains in response to a given stimulus were not similar across individuals, one could call the entire human race delusional since we all go through our everyday lives and interact with others as if they perceive the world in pretty much the same way as we do. As a matter of fact, if the phenomenal world of each individual were unique, the most fundamental social consensus would be lost. Sharing feelings by verbalizing emotions would be an illusion and our use of language as if each word designates the same qualia would be incorrect. It thus appears reasonable to hypothesize that qualia are similar across individuals and that we are actually living in similar phenomenal worlds.\n\nAt first sight, it is tempting to say that qualia could be similar because of the resemblances existing between the brains of humans. However, this idea is questionable for several reasons. First, when macroscopically comparing the brain of people, one can be stricken by the large differences existing between their shapes (with some extreme, such as the one described by Feuillet et al.7). There are also problems at the microscopic level. For instance, nothing has been found that distinguishes the so-called color-cells of V1 for blue from the V1 color cells for yellow apart from their afferences8. Thus, applied within a person, the neuronal similarity argument would predict that qualia for blue should be similar to the qualia for red or yellow. Another point can be made with the qualia for white, which is generated by the stimulation of the three types of cone cells. Or even by only two complementary colors (e.g., green and red, which stimulate the M and the L cone cells, or blue and yellow). How could the V1 “color cells”, which are processing the output of these cone cells generate the same qualia? There again, similarities between particular neurons and qualia do not work. So the hypothesis of a similarity of qualia creates a problem. How could qualia be similar across individuals when they are said to be, by nature, totally private events not strictly dependent on brain similarities?\n\nAnother, apparently unrelated, question is: how can qualia within a given person be so qualitatively different from one another while theoretically originating from the same type of neuronal bioelectrical activity? Sounds appear to be totally orthogonal to colors or smells. Nevertheless, they are induced by the same depolarizations, such as those induced by Penfield and Jasper9 at different places of the cortex. One way to answer this question is to hypothesize that, while dependent on the well-known bioelectrical activities of neurons, the physical nature of qualia is not limited to these activities. The authors of this second hypothesis can grossly be divided into those suggesting, (a) that qualia are also electromagnetic fields (for a recent review, see Jones10) and (b) those developing the even more controverted theory that qualia also include modulations of the wave function described by quantum mechanics (e.g., 11). Each of these two theories thus introduces phenomena, which, by the immense variety of the instances they include, could provide ways to account for the qualitative differences existing between percepts.\n\nInterestingly, thinking about qualia in terms of electromagnetic fields or in terms of modulations of the wave function could also provide a hint as to how qualia are apparently projected to form our perceived environment and also how they could be similar across individuals while being “private events”. Indeed, both physical phenomena propagate. They can thus be projected and travel between individuals. Therefore, some kind of inter-subjective sharing could theoretically occur. In other words, experiencing a qualia might have an impact on the qualia of another person. This means that, at least in some conditions, the brain activity of a person might be influenced by the activity of the brain of another person. No study has reliably12 reported such a direct brain to brain impact but that might be due to the fact that, to the best of our knowledge, no author has yet specifically explored the possibility that qualia propagate.\n\nTesting this possibility was the first aim of the present study. To achieve this goal, we focused on the centro-parietal P600, a late event-related brain potential (ERP) elicited by the presentation of meaningful stimuli, such as, words, objects, faces and scenes. This component belongs to the P3b family of components despite its late maximum, which occurs around 600 ms post stimulus onset when using complex stimuli such as words, objects, faces and even a little later when using scenes. Its amplitude has been reliably related to conscious perception. The greater the amount of information placed in working memory, the larger the amplitude of this potential13,14. Meaningful stimuli presented during attentional blinks that are not consciously perceived elicit no P600, whereas these stimuli do so systematically when they are consciously perceived15,16. On the contrary, the negative component that precedes it, namely, the N400, is evoked by these stimuli even when their processing is only preconscious15–17. Our first goal was thus to measure the amplitude of the P600 elicited by the presentation of a meaningful stimulus to a subject and see if it could depend on whether another person is simultaneously presented with the same stimulus or with a different one (for our purposes, the sameness factor) when one person could not see what the other was presented with. One, potentially greater, impact on the P600s was predicted in the case where different stimuli were presented to each subject, given that the qualia corresponding to each stimulus would be different, and thus that they would generate a greater amount of information in working memory. A different, potentially smaller, impact on the P600s was foreseen in the cases of identical stimuli, since qualia of each person would be similar. An ERP difference between these sameness conditions would support propagation of qualia from the brain of a person to the brain of another person. If this difference pertains to the P600, it would also provide a strong argument for the possibility that the qualia of one person can impact that of another person since qualia are defined here as the building blocks of consciousness and since P600s index consciousness13–16.\n\nHowever, if the brain activity of one person could have an impact on the brain activity of another person, it seems that this impact should be prevented as much as possible when it is known that the other person is confronted with a different stimulus. Indeed, in these conditions, it seems that the qualia of that person should not interfere. The second aim of the present study was thus to manipulate the beliefs of each pair of participants (for our purposes, the belief factor) by telling them that they would be presented with the same stimuli in some conditions and with different stimuli than their partner in other conditions. These statements were true in half of the blocks and false in the other half, while, again, participants had no way to check and no reason to doubt the statements. Our operational hypothesis was that the hypothesized impact of one participant of a pair on the amplitude of the P600 of the other participant would be minimal when participants are presented with different stimuli and believe it. This was used as our baseline condition.\n\nThe third aim of the study had no relation whatsoever to the exploration of the causes of the assumed similarity of qualia across individuals. It was totally separate from the possibility of an impact of one’s activity on the brain of another person. This third goal was to evaluate the impact of social cognition on memory. Indeed, having a mental representation of a partner going through an event (i.e., the presentation of a stimulus), in addition to having a representation of oneself going through the same event, might enrich the encoding in episodic memory and facilitate delayed recognition. Thus, subjects were told to remember each image because there would be a memory test at the end. Our operational hypothesis was that they would have a higher rate of recognition for the stimuli they were presented with when they believed they were seeing the same stimuli as their friend and a lower rate of recognition for the other stimuli.\n\n\nMethod\n\nThirty-two right-handed participants (25 F, 7 M), pairs of friends, couples, or siblings were recruited because it was assumed, for this first attempt, that testing people in a close relationship could increase the odds of positive findings. The 32 subjects of the 16 pairs underwent exactly the same procedure. All participants learned about the experiment through classified ad websites. They spoke fluent English, were between eighteen and thirty years of age (mean = 23.1, SD = 3.4) and had completed, or were in the process of completing, a university degree. They had normal or glasses-corrected to normal vision. Participants were excluded if they consumed more than twelve drinks of alcoholic beverages per week or if they used recreational drugs, except if they used marijuana less than once per week. Participants were also excluded if they had a history of psychiatric disorder, took medication related to such a disorder, or if one of their first degree relatives had a history of schizophrenia or bipolar disorder. All these inclusion- and exclusion-criteria were checked by an eligibility questionnaire.\n\nThe two participants of each pair came to the lab together for approximately three hours. Each participant read and signed an informed consent form accepted by the Douglas Institute Research and Ethics Board. This board, which follows the principles expressed in the declaration of Helsinki, also approved the study itself (Douglas REB #12/12). Data were anonymesed, which did not distort scientific meaning.\n\nStimuli were images selected from the International Affective Picture System (IAPS,18). Using our own judgment, we chose the 560 most striking pictures of this set to ensure the maintenance of participants’ attention during the tasks. The experiment consisted first of the study phase, which included four blocks, and of a memory test phase. As presented in Table 1, which explains their acronyms, each of the four blocks of the study phase, DBd, SBs, SBd and DBs, corresponded to a particular sameness and belief condition. The order of presentation of these four blocks was randomized across subject pairs using a Latin square. We used four different sets of 70 IAPS stimuli. The allocation of each set to each block was also randomized across subject pairs. In study phase blocks in which different pictures were seen by each member of a pair (i.e., in the DBd and DBs blocks), the picture set seen by one participant in DBd was seen by the other participant in DBs, and vice versa. Therefore, all pictures of the four sets were seen by both participants during the study phase. The memory test phase consisted of a fifth set of pictures that contained, in a random order, all the pictures of the study phase mixed with 280 additional pictures.\n\nThe study phase (Table 1) was followed by the memory test phase. As illustrated by Figure 1, each stimulus of the study phase was presented for 1000 ms and was followed by a white screen with a black fixation cross, the duration of which randomly varied between 790 and 1500 ms to prevent the development of a contingent negative variation19. Participants could see their partner in their very peripheral vision field without moving their eyes. Nevertheless, even if they moved their eye or did head movements, they could not see the part of the screen their partner was watching (Figure 2 illustrates this unusual setting). Participants were told to look at each picture for the subsequent memory test phase. Stimuli in that latter phase were presented for 3000 ms in order to allow time for participants to respond.\n\nThe different-and-believed-different (DBd) condition is used as an example. Note the division of the screen into two halves by the vertical cardboard piece, preventing the two subjects from seeing each other’s stimuli, but not preventing them from feeling close to one another.\n\nDuring the memory test phase, participants were required to respond by pressing keys on a shared computer keyboard. The participant seated on the left hand side of the keyboard used the typewriter keys and pressed ‘1’ to indicate (s)he believed to have seen the picture previously, and ‘2’ to indicate (s)he believed not to have seen the picture previously. The participant seated on the right hand side of the keyboard used the numeric keypad and pressed ‘4’ to indicate (s)he believed to have seen the picture previously, and ‘5’ to indicate (s)he believed not to have seen the picture previously.\n\nAt the end of the memory test phase, there was a debriefing session where participants were asked 4 questions, mainly designed to explore attention differences and whether they detected any deception. The first was: “Did you feel more attentive/distracted seeing the pictures when your friend was present?”. The second was: “Did you feel any different when you knew your friend/partner/relative was looking at the same images that you were seeing?”. The third was: “Did you feel any different when you knew your friend/partner/relative was looking at different images than you were seeing?”. The fourth was: “Did you feel deceived at any point during the experiment?”.\n\nBehavioral key presses were recorded during the memory test phase, as well as the verbatim of the response to the debriefing session’s questions. The electroencephalogram was recorded from 28 electrodes mounted in an elastic cap (Electro-Cap International) during the study phase. Electrodes were placed according to the modified expanded 10–20 system20. For each participant of each pair, these electrodes were grouped into three subsets: sagittal (Fz, Fcz, Cz and Pz), parasagittal (Fp1/2, F3/4, Fc3/4, C3/4, Cp3/4, P3/4, and O1/2), and lateral (F7/8, Ft7/8, T3/4, Tp7/8 and T5/6). There was a separate set of amplifiers for each participant. The right earlobe was used in each subject as the reference for his/her set of amplifiers while the ground of each participant was taken from an electrode two centimeters ahead of Fz. For both sets of amplifiers, high- and low-pass filter half-amplitude cut-offs were set at 0.01 and 100 Hz, respectively, using an additional 60 Hz electronic notch filter. EEG signals were amplified 10,000 times and digitized online at a 256 Hz sampling rate and stored in a single file with 56 (28 × 2) channels.\n\nIn each trial, electrodes contaminated by eye movements, excessive myogram, amplifier saturations or analog to digital clipping were removed offline by setting automatic rejection criteria. Electrodes for which analog to digital clipping exceeded a 100 ms duration and electrodes for which amplitude exceeded +/- 100 mV were discarded. Before these rejections, the baseline was set prior to the onset of the stimulus, from -200 to 0 ms. Averages were calculated for each block and each subject in a 1400 ms time window, beginning 200 ms before the onset of the stimulus and lasting for 1200 ms after the stimulus onset. Following averaging, each file was divided into two files, each containing the ERPs of a single subject. The ERPs of each of the 32 subjects were then computed and measured independently of the pair of participants they initially belong to. Based on our a priori hypothesis, we focused on the late positive component (LPC or P600) and computed the mean voltages of ERPs in the 600–900 ms time window for all electrodes, all conditions and all subjects.\n\nThree repeated-measures ANOVAs were run with the version 20 of the IBMSPSS software package to analyze these measures using a multivariate approach. They had sameness (same vs. different stimuli), belief (belief that stimuli were the same vs. belief they differed) and electrodes as within-subject factors. For parasagittal and lateral electrodes, a fourth within-subject factor, hemiscalp (right vs left), was included. Given that there was only one group of 32 subjects, there was not any between-subject factor. Post-hoc analyses were completed for interactions whose p values were smaller than 0.1. The Greenhouse and Geisser21 procedure was used when required to compensate for heterogeneous variances, in which case the original F values and the corrected p values will be given. To provide a priori hypotheses for future studies, we also completed one-way ANOVAs at each electrode to assess each effect found.\n\n\nResults\n\nFigure 3 shows the grand averages for the 32 subjects of the 16 pairs tested. Visual inspection of the P600 time window at the electrodes where the amplitude of this ERP component is usually maximal, that is, at the central (Cz) and parietal (Pz) midline sites, reveals that the smallest P600s were obtained for the baseline condition of the study phase where stimuli were different and where participants believe they were seeing an image different from that presented to the other member of their pair (the DBd condition, with the blue waveforms in Figure 3). P600s appear a little bit larger for the Same Believe-same condition (SBs, green waveforms) and maximal for the different believe-same (DBs, orange) and the same believed different condition (SBd, red).\n\nBlue waveforms are for the condition where the two stimuli were different and were believed to be different (DBd); green for when they were, and were believed to be, the same (SBs); orange: different stimuli but believed to be the same (DBs); red, same believed-different (SBd).\n\nTable 2 includes the F and p values of the ANOVAs performed on each subset of electrodes.\n\nTable 3 contains the results of the post-hoc analyses run for each electrode subset to explore the sameness × belief interactions reported in Table 2.\n\nIn addition to the findings presented in Table 2 and Table 3, a significant belief × hemiscalp interaction at the lateral electrode set prompted a further analysis, which revealed a marginally significant effect of belief over the left hemiscalp, F(1-31) = 4.24, p = .05.\n\nEffects were then explored relative to the condition where stimuli were different and believed to be different (DBd), as this was the condition where the smallest impact of others’ qualia should occur. Spline interpolated isovoltage scalp maps, including the p values for each electrode (Figure 4), were built to illustrate the scalp distribution of the differences from that baseline condition. These maps were thus made by subtracting the mean voltages of the ERPs in the 600–900 ms time window of that baseline condition (e.g., the DBd condition, blue curves) from those of another condition (e.g., the SBd condition, red curves) at each electrode sites. Although, note that, according to the Bonferroni correction, only 4 stars-sites would be significant when considering electrodes other than Cz and Pz. For the first two maps (Figure 4) the other conditions were SBd and to DBs, respectively. These two maps show that the differences were significant at a large number of scalp sites. In contrast, the 3rd map reveals that the differences between SBs and DBd were more localized at left frontal sites.\n\nSpline interpolated isovoltage scalp maps computed by subtracting mean voltages of the 600 to 1000 ms time window. P values of the differences are indicated at each electrode site by stars. The baseline condition (i.e., different & believed-different, DBd) was subtracted, in A) from the same & believed-different (SBd) condition, in B) from the different & believed-same (DBs), and in C) from the same & believed-same condition (SBs). Note that, according to the Bonferroni correction, only 4 stars-sites would be significant when considering electrodes other than Cz and Pz since the alpha level would be 0.0018.\n\nOn the other hand, the replicability of these findings was explored by computing grand averages of the 16 subjects of the first 8 pairs and the grand averages of the 16 subjects of the last 8 pairs of participants separately. Note that these two sets of subjects went through the exact same procedure and conditions. Figure 5 and Figure 6 display these grand averages. At Cz and Pz, the amplitudes of the P600s for each condition appear to be in the same increasing order, that is from DBd (blue) to SBd (red), via SBs (green) and DBs (orange), as in the grand averages of the 32 participants presented in Figure 3.\n\nColors as in Figure 3.\n\nColors as in Figure 3 & Figure 5.\n\nWe also explored whether large differences in one of the member of the pair were going with large differences in the second member while small differences in one member were going with small differences in the other member. We focused on the conditions that were the most different from each other, namely, SBd-DBd and DBs-SBs and computed the correlations between subjects of each pair for each electrode in order to generate a priori hypotheses for future studies. The significant results that were found are presented in Table 4. Meanwhile, Figure 7 presents scatterplots made by using the data for the electrodes most relevant for the P600, that is, the parietal electrodes P3 and P4 where maximal correlation coefficients were obtained. These correlation coefficients (Table 4) show that, when participants believed they were seeing the same pictures (Bs), the larger the effect of sameness in one participant, the larger this effect in the other. In contrast, when they believed they were seeing different pictures (Bd), these correlations were negative (top of Table 4).\n\nAll the correlations having a p value smaller than 0.05 are indicated to generate a priori hypotheses for future studies. However, using a Bonferroni correction for doing 28 analyses (one for each electrode), leads to a corrected alpha level of 0.0018.\n\nThe x coordinate of each point is the size of the P600 effect in one participant of a pair and the y coordinate of the point is that size for the other participant of that pair. At left parietal site (P3) in the believe-different conditions (SBd-DBd), the graph reveals that the greater the size of the effect of sameness on the P600 amplitude in one participant, the smaller this effect in the other member of the pair. In contrast, at right parietal sites (P4) in believe-same conditions (DBs-SBs), the greater the effect in one participant, the greater this effect in the other. The correlation coefficients for these electrodes, as well as for others, are presented in Table 4.\n\nAs shown in Table 5, in the memory test phase, there was no difference between study phase conditions in the number of stimuli correctly recognized (hits) or in the number of misses. In sum, participants did not better recall images from any particular condition of the study phase. Similarly, there was no effect of the condition of the study phase on the reaction times of the memory test phase (Table 6).\n\nThe results of the debriefing session were as follows. For the question: “Did you feel more attentive/distracted seeing the pictures when you friend was present?”, 8 participants said they were more distracted, 18 said there was no difference, 6 said they were more attentive. To the question: “Did you feel any different when you knew your friend/partner/relative was looking at the same images that you were seeing?”, 17 participants said they felt the same, 14 said they felt different. To the question: “Did you feel any different when you knew your friend/partner/relative was looking at different images than you were seeing?”, 9 said yes, 22 said no. For the fourth question “Did you feel deceived at any point during the experiment?”, 27 said no, 3 misunderstood “deceived”, 2 said yes, but when asked why, they did not suspect the statements of sameness of stimuli. Their suspicion pertained to other aspects (e.g., one said, after the stimulus presentation computer unexpectedly stopped, “I thought that when the computer crashed it was deliberately done so that it was more difficult to remember”).\n\n\nDiscussion\n\nIn each recording session of this study, pairs of related participants were tested together. In each trial, two pictures taken from the international affective picture system (IAPS) were presented simultaneously, one for the first participant, the other for the second participant of the pair. All 32 participants of the 16 pairs tested were asked to remember these pictures during the four different blocks of the study phase. These pictures were then presented again, mixed with new ones, during a subsequent memory test phase.\n\nDuring both phases, the computer screen was divided in two halves that were separated by a vertical cardboard perpendicular to the screen. Each participant of a pair sat in front of one half of the screen and was presented with one picture at a time. There was no way for a participant to see the picture simultaneously presented to the other participant.\n\nSameness was manipulated. In two of the four conditions of the study phase, participants were presented with the same picture simultaneously (S conditions). They were presented with two different pictures in the two others conditions (D conditions). The belief (B) pertaining to what the other member of the pair was presented with was also manipulated. Just before the beginning of each condition, or block, of the study phase, participants saw one of two statements on the screen, announcing whether or not the same (Bs vs. Bd conditions) the same picture would appear for both of them on each half of the screen. The four conditions of the study phase were thus: different believed-different (DBd), same believed-same (SBs), different believed-same (DBs) and same believed different (SBd), the latter two thus including deceiving statements.\n\nEvent-related brain potentials elicited by the IAPS pictures were recorded during these four conditions of the study phase. There was an interaction of sameness with belief on the amplitudes of the P600s. In the believed-different conditions (Bd), these amplitudes were significantly larger when pictures were actually the same (SBd) than when they were different (DBd), as illustrated by Figure 3 and Figure 4. In contrast, in the believed-same conditions (Bs), P600 amplitudes tended to be larger when pictures were different (DBs) than when they were the same (SBs).\n\nUsual interpretations of these ERPs difference could be ruled out. First, because, in contrast with the third hypothesis, there was no effect of sameness or belief on the recognition scores obtained during the memory test phase. The ERP differences found between the different conditions of the study phase could thus not be related to a Dm effect; that is, to larger P600s at fronto-central electrode sites for stimuli that benefit from a deeper encoding in episodic memory22,23. Second, the ERP differences found were also unlikely to be related to differential allocations of attentional resources. Indeed, all stimuli had the same task relevance since they equally had to be memorized. Moreover, they could not capture attention differentially, since they were identical because their use for each of the conditions of the study phase was counterbalanced across pairs of participants.\n\nThe statements seen by participants as to whether or not they would be presented with the same stimuli as the other participant of the pair could have theoretically modulated the allocation of attentional resources and thus P600 amplitudes. Nevertheless, these statements could not have had an effect depending on the actual sameness of the stimuli, since it was something participants had no knowledge of. Third, more preconscious processing does not seem to be useful to account for the greater P600s obtained for the three conditions other than DBd. Indeed, why would more processing have occurred for these SBs, SBd and DBs conditions than for this baseline condition when all stimuli equally had to be memorized?\n\nOn the other hand, participants were side by side and could get some auditory and visual input from each other in their very peripheral field (i.e., 90 degrees). Thus, they could in principle influence each other (e.g., through breathing variations, subtle body movements, like postural reactions to aversive stimuli, facial mimicry, eye movements etc). It thus has to be discussed whether or not the present results could be in line with Dumas’ et al. work24 on hyperscanning and inter-brain synchrony mediated through the mirror neuron system. Indeed, direct brain-to-brain propagations do not appear to be the most parsimonious explanation. Given that our participants did not have any task to perform, other than to look at the stimuli, part of their attention could have been allocated to what their friend was doing. Therefore, we have to ask whether the processing of these movements could have been responsible for our results. Nevertheless, for ERPs to differ across conditions in a systematic way, as they did in the present experiment, the movements (or breathing sounds) made by the friend should depend on whether or not the stimuli (s)he was presented with are the same as the one the subject is seeing. This does not seem impossible since, when participants were not seeing the same stimuli, they might not “be moved” in the same way. Their systems might have detected that move difference. However, to account for the results obtained here, the effect of such a detection would also have to depend on whether or not the subject was told that (s)he was presented with the same stimuli as his friend. When (s)he has been told stimuli differ (as in the DBd condition) the move difference detected is congruent with the statement. When the subject was told (s)he will be presented with same stimuli, then, the move difference detected should be further processed since it is contradictory information. However, ERP results are not consistent with this interpretation. Contradiction or incongruence is well known to boost the amplitude of negative going ERPs, such as the N2 and the N400 [e.g., 25, 26]. It thus has an effect on potentials other than the P600 and in the reverse direction. This is completely discordant with the present results. And, even if we hypothesize a very unusual ERP whereby greater P600s would index more processing difficulty, the account would then not explain why the P600s elicited by SBs appears larger than the one elicited by DBd, whereas, in that condition, statement and stimuli were congruent.\n\nTherefore, in accordance with the first two hypotheses, the fact that P600s were larger than DBd in three conditions other than DBd and that P600 amplitudes correlate positively with consciousness13–16 suggest that the two participants of each pair may actually enrich the content of conscious awareness of one another. These effects suggest that the activity of the brain of a participant may have a direct impact on the activity of the brain of the other participant. Given, that the P600 component also indexes conscious perception13–16, these results could thus be related to qualia, the individual instances of subjective conscious experience.\n\nOn the other hand, because only phenomena that propagate can account for the impact of one brain on another, these results also suggest that qualia are not limited to the known bioelectrical activity of neurons. They may also include physical phenomena of a different nature, such as electro-magnetic fields, as reviewed by Jones10, or such as modulations of the so-called wave function, studied in quantum mechanics and debatably proposed by Hameroff and Penrose11. The electromagnetic hypothesis can be based on the sensitivity to magnetic fields of at least two molecules: magnetite, whose presence has been demonstrated in the human brain27–29, and cryptochrome30. Furthermore, it is consistent with the fact that mammal behaviors have been shown to depend on magnetic fields, such as that of the earth31,32. However, two properties of magnetic fields are at odds with the idea that the magnetic fields generated by one participant could affect the brain activity of the other participant. First, the magnetic fields generated by the activity of the human brain (only 10 to 103 femto Tesla) are much smaller than the magnetic noise of an urban environment (about 108 femto Tesla). Second, magnetic fields decrease with the square of the distance. The heads of the two subjects of each pair were separated by about 40 cm, a distance much larger than the distance separating the brain from the devices used to capture the magnetic fields it generates in magneto-encephalography (MEG, i.e., less than one cm). Finally, our ERP recording room was not shielded like a MEG recording room. Urban magnetic noise was thus much more important than any field a human brain can generate. These factors make the electromagnetic field explanation appear less likely. In contrast, our experimental conditions and results seem to be more consistent with the theories of consciousness that see qualia as, at least partly, underlain by a modulation of the wave function, and that see direct brain to brain communications possible through quantum entanglement33. Indeed, such modulations do not decrease with distance and could involve many atoms34. Nevertheless, only speculations can be made at this point as to the physical nature of the phenomena by which the activity of a brain could have an impact on the activity of another brain.\n\nThe finding of such an impact raises the problem of irrelevant interferences. Indeed, the activity of many brains could then affect the activity of our own. It appears logical to think that filtering exists to prevent such perturbations. One possibility is that, the close relationship existing between the members of each pair in the present study is a prerequisite for the impact to occur, as it may depend on empathy and/or prior common memories. On the other hand, filtering should operate to a greater extent when it is believed that others are processing different stimuli. The results of the present study suggest that this might be the case. When participants were told that they would be presented with different stimuli, the P600s were minimal, which was taken as the baseline condition. However, this happened only when they were actually presented with different stimuli. In the case where the two stimuli were the same (SBd), the P600s were maximal, suggesting that this “belief-based filtering” can operate only when qualia actually differ. P600s at Cz were also maximal when participants believed they were seeing the same stimuli while different ones occurred (DBs). Notably, the scalp distribution of these two additional P600 activities differs from the scalp distribution of the additional P600 activity found when comparing SBs to the baseline condition (DBd) (Figure 4). The latter appeared localized at left frontal sites whereas the former two included that location but were also widespread. This latter fact could suggest that while the “enrichment” of consciousness occurred also in deception conditions, the evaluation of its coherence with the belief might bring up yet additional content in consciousness.\n\nThe fact that, at left frontal sites, the 600–900 ms time window used was mainly including the downhill slope of a negativity starting much earlier may be important. Rather than smaller P600s, the significant effects found at these electrode sites might in fact reveal larger late N400s for stimuli that were, and were believed to be, different. This change of perspective might provide an a priori hypothesis for future studies of the filtering mechanism proposed above. Indeed, the N400 has been proposed as an index of an inhibition mechanism whose focus depends on the nature of the inhibited representations [for a brief review see 17].\n\nOn the other hand, the nature of the enrichments suggested by the greater midline P600s has to be discussed. The fact that no deception was detected, that is, that no subject realized that (s)he was looking at different stimuli when told (s)he was looking at the same, suggest that the additional content of consciousness was neither verbalizable nor distinguishable from the qualia each participant would have had if (s)he were alone. This strongly supports the mutual enrichment hypothesized in the introduction, where qualia of others would contribute to our own by a merging process occurring without our knowledge.\n\nInterestingly, when participants were told the same stimuli were appearing, the effect of sameness on the size of the P600s in one of the members of a pair positively correlated with that size in the other member (Table 4 & Figure 7). In other terms, the greater the effect in one person, the greater the effect in the other. On the contrary, when participants were told different stimuli were appearing, the correlation was negative, as if the greater the effect in one person, the more its impact was detected and could be prevented in the other.\n\nThere is a tradition of research studying the synchronization of EEGs and bold fMRI signals of two persons interacting, imitating each others’ movements [e.g., 35] and of persons going through the same stimulation(s) [e.g., 36, for a review, see 37]. This tradition could be relevant here since, we also recorded the EEG of two participants simultaneously. However, we used ERPs, not EEGs’ synchrony or fMRI, and our participants were not interacting, imitating each other, or being presented with only the same stimulation. Each subject in a pair was going through the experiment on his/her own “despite” the fact that (s)he was sitting side by side with a friend/sibling/spouse. Sameness, and belief in that sameness, were manipulated, which modulated the amplitude of an well-known ERP index of consciousness. To the best of our knowledge, there is thus yet no equivalent to the present study. The hypothesis of a direct sharing of qualia has never been tested. Future studies have to explore whether differential EEG synchrony can also occur within the present design and also test whether qualia sharing could account for part of the EEG synchrony observed in interacting participants, for instance. Indeed, the conscious intention to perform an action, when imitating, can be considered as a qualia and could, according to the present results, impacts the functioning of the brain of the interacting person.\n\nIt has to be noted that, if further replicated, these findings could open several avenues of research. For instance, it might be interesting to explore whether young children’s brains learn to produce their qualia with the help of others. It could also be interesting to see if autistic children suffer from a disability of this learning mechanism or whether their tendency to limit contact with others is a strategy that protects them against a deficit of the filtering mechanism.\n\nIn any case, the results of the present study provide preliminary data about the mechanisms by which qualia pertaining to the same stimulus could be similar across individuals, something that is assumed in everyday life interactions. Results also suggest that the similarity could be due to an intersubjective impact of brain activities, which could be partially controlled and whose physical bases would remain to be determined.\n\n\nData availability\n\nF1000Research: Dataset 1. Mean voltages of the event-related brain potentials elicited by the IAPS stimuli., 10.5256/f1000research.5977.d4121538", "appendix": "Author contributions\n\n\n\nJ. Bruno Debruille wrote the research project that was accepted by the Research and Ethics Board of the Douglas Institute, designed the experiment, interpreted the data and wrote the introduction and the discussion of the manuscript. Sheila Bouten built the stimulus sequences, recruited the participants, tested them, analyzed the data, took part in their interpretation, wrote the method and the result section of the paper and proof read all the preliminary versions of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by the grant 194517-03 from the Natural Sciences and Engineering Research Council of Canada allocated to the corresponding author.\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\nAuthors thank Tara Campbell for running a preliminary version of the experiment. She also helped for the revised submission, as well as Daniel Ramirez Rodriguez.\n\n\nReferences\n\nSearle JR: How to study consciousness scientifically. Philos Trans R Soc Lond B Biol Sci. 1998; 353(1377): 1935–1942. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDennett DC: Quining Qualia. In Consciousness in contemporary science. A J Marcel & E Bisiach Eds. Oxford Science publication, Clarendon Press Oxford. 1988; 43–77. Reference Source\n\nPetitot J, Smith B: Physics and the phenomenal world. In R. Poli and P. M. Simons (eds.), Formal Ontology, Dondrecht/Boson/London: Kluwer. 1997; 223–254. Reference Source\n\nEhrsson HH: The experimental induction of out-of-body experiences. Science. 2007; 317(5841): 1048. PubMed Abstract \| Publisher Full Text\n\nLakoff G, Johnson M: Metaphors we live by. Chicago, Illinois, University of Chicago Press. 1980. Reference Source\n\nGiora R: Understanding ﬁgurative and literal language: the graded salience hypothesis. Cognitive Linguistics. 1997; 8(3): 183–206. Publisher Full Text\n\nFeuillet L, Dufour H, Pelletier J: Brain of a white-collar worker. Lancet. 2007; 370(9583): 262. 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PubMed Abstract \| Publisher Full Text\n\nLang PJ, Bradley MM, Cuthbert BN: International affective picture system (IAPS): Technical manual and affective ratings. The Center for Research in Psychophysiology, University of Florida, Gainesville, FL. 1997. Reference Source\n\nTecce JJ: Contingent negative variation (CNV) and psychological processes in man. Psychol Bull. 1972; 77(2): 73–108. PubMed Abstract \| Publisher Full Text\n\nElectrode position nomenclature committee: American Electroencephalographic Society guidelines for standard electrode position nomenclature. J Clin Neurophysiol. 1991; 8(2): 200–202. PubMed Abstract \| Publisher Full Text\n\nGeisser S, Greenhouse GW: On methods in the analysis of profile data. Psychometrika. 1959; 24(2): 95–112. Publisher Full Text\n\nFriedman D, Trott C: An event-related potential study of encoding in young and older adults. Neuropsychologia. 2000; 38(5): 542–557. PubMed Abstract \| Publisher Full Text\n\nPaller KA, Kutas M, Mayes AR: Neural correlates of encoding in an incidental learning paradigm. Electroencephalogr Clin Neurophysiol. 1987; 67(4): 360–371. PubMed Abstract \| Publisher Full Text\n\nDumas G, de Guzman GC, Tognoli E, et al.: The human dynamic clamp as a paradigm for social interaction. Proc Natl Acad Sci U S A. 2014; 111(35): E3726–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAppelbaum LG, Smith DV, Boehler CN, et al.: Rapid modulation of sensory processing induced by stimulus conflict. J Cogn Neurosci. 2011; 23(9): 2620–2628. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKutas M, Van Petten CK, Kluender R: Psycholinguistics electrified II. In M.A.Gernsbacher & M.Traxler (Eds.), Handbook of Psycholinguistics, 2nd edition. New York: Elsevier Press. 2006; 659–724. Publisher Full Text\n\nDobson J, Grassi P: Magnetic properties of human hippocampal tissue--evaluation of artefact and contamination sources. Brain Res Bull. 1996; 39(4): 255–259. PubMed Abstract \| Publisher Full Text\n\nDunn JR, Fuller M, Zoeger J, et al.: Magnetic material in the human hippocampus. Brain Res Bull. 1995; 36(2): 149–153. PubMed Abstract \| Publisher Full Text\n\nKirschvink JL, Kobayashi-Korschvink A, Woodford BJ: Magnetite biomineralization in the human brain. Proc Natl Acad Sci U S A. 1992; 89(16): 7683–7687. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolov’yov IA, Schulten K: Reaction kinetics and mechanism of magnetic field effects in cryptochrome. J Phys Chem B. 2012; 116(3): 1089–99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurda H, Begall S, Cervený J, et al.: Extremely low-frequency electromagnetic fields disrupt magnetic alignment of ruminants. Proc Natl Acad Sci U S A. 2009; 106(14): 5708–5713. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHart V, Nováková P, Malkemper EP, et al.: Dogs are sensitive to small variations of the Earth’s magnetic field. Front Zool. 2013; 10(1): 80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllen M: Nothing spooky about decoding telepathy: a lesson in the value of open science by Micah Allen. PLoS One. 2014. Neuroscience Community, Comment posted on Oct1st, 2014. Reference Source\n\nWeidenmüller M: Quantum physics: spooky action gets collective. Nature. 2013; 498(7455): 438–439. PubMed Abstract \| Publisher Full Text\n\nBurgess AP: On the interpretation of synchronization in EEG hyperscanning studies: a cautionary note. Front Hum Neurosci. 2013; 7: 881. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHasson U, Nir Y, Levy I, et al.: Intersubject synchronization of cortical activity during natural vision. Science. 2004; 303(5664): 1634–40. PubMed Abstract \| Publisher Full Text\n\nHasson U, Ghazanfar AA, Galantucci B, et al.: Brain-to-brain coupling: a mechanism for creating and sharing a social world. Trends Cogn Sci. 2012; 16(2): 114–121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBouten S, Debruille JB: Mean voltages of event-related brain potentials elicited by the IAPS stimuli. F1000Research. 2014. Data Source" }	[ { "id": "7149", "date": "07 Jan 2015", "name": "André Achim", "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 scientific study of consciousness is extremely difficult. Any serious attempt should therefore be regarded with a positively open mind. The intellectually challenging results of the present experiment, however, are unlikely to pertain to understanding the nature of qualia in the human brain. They rather seem to hint at a social cognition effect.DesignThe data consist in the average amplitude from 600 to 900 ms post stimulus onset of event related potentials (ERP) to complex stimuli selected as “most striking pictures” from the International Affective Picture System (IAPS). EEG recordings were obtained from dyads of socially related participants, simultaneously presented on separate halves of the same screen with a picture that could not be seen by the other participant, even though they were seated side by side and could see each other in the periphery of their visual field. Each participant had to observe the pictures in order to recognise them in a later memory test.Four different ERP conditions are compared, each consisting of the average of 70 stimuli (minus occasional artefacts at some channels) obtained from the same block. Two of the blocks were introduced to the two participants to consist of identical stimuli simultaneously presented to each of them, and the remaining two blocks were announced to consist of different sets of pictures. In each pair of conditions, this initial announcement was faithful once and contrary to the actual situation the other time. This allowed analyzing the situation in a 2 x 2 design of Sameness (Identical versus different pairs actually presented) by Belief (believing that the partner was presented with same versus different stimuli).The recordings, in each participant, were from 28 channels, referred to right ear lobe, not including any EOG dedicated channels. Each picture was presented for 1.0 s with a mean expected inter stimulus interval just below 1.15 s. Therefore, each of the four stimulus block lasted only about 2.5 minutes. The following simultaneous testing phase of the two participants required 28 minutes (280 targets + 280 new items each presented for 3.0 s).Although the rationale for the study considers qualia and the possibility of common brain signatures, the averaging over different stimuli in the same condition restricts considerably the relevance of the data to the original question of the nature of the qualia and of their physical support in the brain. The authors actually need to extend the concept of qualia to include the knowledge of whether the other person in the dyad is supposed to receive identical or different stimuli, which is very unlikely the dominant subjective experience (qualia) produced by any stimulus. The data are therefore much more likely to pertain to social cognition than to the intended exploration of a physiological substrate for the qualia. Actually, for features of visual perception that do not play a role in the task (for instance by being rare versus frequent events or relevant versus irrelevant), the ERP differences are likely to be idiosyncratic, requiring special statistical technique to detect differences that take different forms in different brains (e.g. Buchsbaum and Fedio, 1969, 1970, for the ERP difference between geometrical patterns and three letter words made of the same number of dots).ERP results.Visual inspection of the averages shows that the ERP were negative, relative to the 200 ms baseline, for the full duration of the stimuli in all four conditions for all 12 channels anterior to Cz, and were entirely positive in 6 of the seven posterior channels, at the level of, or posterior to Pz, excluding Pz itself which is mostly negative up to about 400 ms and positive thereafter in all four conditions. Despite the large anterior-posterior inversion in overall ERP polarity, the amplitude differences between conditions remain constant in polarity across all channels.The differences in condition are most marked between the two Believe Different conditions, with the Actually Same condition (SBd) being positive relative to the Actually Different condition (DBd). In the right hemisphere and posteriorly in the left hemisphere, the two Believe Same conditions (Actually Same and Actually Different, respectively SBs and DBs) were of intermediate amplitudes. In the frontal half of the left hemisphere, however, the ERP to the two Believe Same conditions overlap substantially with those of SBd, all three being less negative than DBd.The lack of inversion of the condition differences suggests that the underlying dominant effect is unlikely a simple modulation of the dominant sources of these ERP. The left frontal distinction from the remaining ordering of conditions could indicate the presence of at least two sources modulated by the experimental conditions.Although the data were analyzed with Belief and Sameness as factors, according to the experimental plan, it is relevant to decide whether the conditions SBs, SBd and DBs differ among themselves. Since the data are available, these could be tested. The following table gives the p value of all effects involving the three level Condition factor (DBd being excluded).\n\neffect\n\nElectrode group\n\nSagittal\n\nPara sagittal\n\nLateral\n\nCond\n\n.774\n\n.699\n\n.863Cond x Hem\n\n.353\n\n.228Cond x Elect\n\n.208\n\n.128\n\n.489Cond x Hem x Elect\n\n.066\n\n.131 Thus, even without correcting for these 10 tests involving Condition, there is no indication of any difference in this group of three conditions. Since there was no condition in which the participants were alone, it cannot be decided empirically whether the effect of watching stimuli in dyads affects essentially the DBd condition or the remaining three conditions.The “operational hypothesis” that DBd would have a minimal impact on the ERPs implies that the topography in that condition is essentially that of the background activity. Since the background activity inverses polarity from front to back, this should also occur in the DBd condition. Since it does not, it becomes difficult to consider de facto DBd as the baseline no-effect condition. It could well be that DBd is the only condition that expresses the social cognition effect apparently present in the data.Although ad hoc, the following explanation may be proposed for the present data in terms of social cognition. In the DBd condition, each partner might be interested in whether the other person is presented with a similar amount of emotion. This would also be so at the beginning of the SBd condition, but the impression would rapidly build that the stimuli, although believed different, would be matched for emotion and therefore this interest in the amount of emotion felt by the other person could fade. This would account for the SDd-DBd difference being the most reliably detected. For the two Believe Same conditions, each partner would not doubt that the other person receives the same amount of emotion, resulting in no difference between these conditions.CorrelationsThe result section also includes correlations calculated for P600 amplitude differences between conditions. These are reported, in Table 4, only for “the conditions that were the most different from each other, namely, SBd-DBd and DBs-SBs”. This justification seems incorrect, given the above table showing no detected difference between SBd, DBs and SBs, and since additional tests of the latter pair does not show any difference at any channel (all p>=.073). Reproducing the scatter plots of Figure 7 confirms that only 14 intact dyads were actually retained (an odd numbered participant followed by the next even number in the data base provided; the rationale for rejecting some participants should have been expressed). The situation, however calls for using the intra class correlation coefficient (ICC) in which no distinction is made as to which member should be A and which should be B, and for which only the common sample mean and common sample variance are used, resulting in one extra degree of freedom for the test, since only one mean is fitted to the data. When ICC are calculated, the following can be obtained (the sign after the channel name duplicates that of the correlation).ICC in SBd-DBd: 4 channels with p<.05\n\n9 r=-0.5674 t(13)=-2.4843 p=0.0274* F8-\n\n17 r=-0.5438 t(13)=-2.3363 p=0.0361* Fz-\n\n20 r=-0.6196 t(13)=-2.8462 p=0.0138* P3-\n\n22 r=-0.6207 t(13)=-2.8545 p=0.0135* Pz-ICC in ICC in DBs-DBd: no channel with p<.05ICC in ICC in SBs-DBd: no channel with p<.05ICC in SBd-SBs: 1 channel with p<.05\n\n21 r= 0.6947 t(13)=3.4823 p=0.0040** P4+ICC in SBs-DBs: 3 channels with p<.05\n\n18 r= 0.5142 t(13)= 2.1617 p=0.0499* O1+\n\n19 r= 0.5305 t(13)= 2.2562 p=0.0419* O2+\n\n21 r= 0.6208 t(13)= 2.8551 p=0.0135* P4+The other condition differences were not tested. Note that 8/140 (perhaps not independent) correlations tested have p<.05, while 7/140 is expected for independent statistical tests when H0 is true. The critical value of p, with a Bonferroni correction (for 140 independent tests) would be .0018, not reached by any of the 140 tests.Besides the possibility of there being no true correlation between the dyad members, the negative correlations between the dyad members, observed for the SBd-DBd difference, are counterintuitive. One could call upon disentanglement in which collapsing of the wave function for a particle causes the collapse of the complementary state, even at great distances. But here we have negative correlations on average amplitude differences. The quantum physics speculation not only would dispose of the phenomenon, assuming it is not a statistical accident, as being apparently explained, but this would require further ad hoc speculations to explain that the wave function would systematically collapse in the same way in the same person in the given test situation.If there is a true phenomenon to understand, we should start by questioning whether the negative correlations are mostly associated with SBd or with DBd. Note that since the same variable is used for both members of the dyads, changing its sign would not alter the direction of the correlation. It is unlikely that DBd is the source of the negative correlations since no significant (p<.05) correlation is seen in any other difference involving DBd. But the source of the negative correlation is not likely to be SBd either since the other tested difference involving SBd does not replicate the negative correlations. While the negative correlations for SBd-DBd were significant (p<.02) at P3 and Pz, it is a positive correlation that is significant (p<.01) at P4 for SBd-SBs. Since P4 also gives a significant (p<.02) positive correlation for SBs-DBs, the positive correlation for SBd-SBs is more likely attributable to SBs than to SBd (which was involved in the negative correlation).Setting aside the problem of ascribing the negative correlation to one of the involved experimental conditions, some important insight about social cognition could come from trying to identify the characteristics which defines which member of a dyad would produce a large or positive difference between conditions and which one would produce small or negative differences. Could this, for instance, characterize an implicit cognitive domination-submission attitude? But whether any of these correlations reflects a true correlation remains to be established first. The above discussion casts serious doubts on this.Technical detailsIn reporting the behavioral data in Table 5, the misses do not need to be reported, as they should be 70 minus the number of hits (not exactly so here probably because of rounding errors), but the false alarm rate for the 280 new pictures should be added to allow estimating the amount of guessing.In Figure 2, it is not clear to what the 20 cm distance applies. It would be relevant, however to know how far apart from each other were the participants and at what distance from the screen were their eyes, so we can appreciate how much they could see of each other. Instead of the first and last half of participants, Figures 5 and 6 could provide the means form the participants on the left and those on the right (even though they are not the same in number), so that any tendency to gaze at the other person at some systematic time after stimulus delivery would be reflected by opposite shifts at F7 and F8. From the data available, there is no systematic group difference if F8-F7 in any or the four conditions, but that tells nothing of the 0-600 ms interval.If systematic correlations existed between dyad members, the independence of participants in the ANOVA would not be achieved, so that the degrees of freedom would actually be inflated. This possible bias could be acknowledged, even though the presence of correlations is not very convincing.On page 6: the statement “electrodes for which amplitude exceeded +/- 100 mV were discarded” would need clarification about the period of time in which this would be observed, since event exclusion was done channel by channel. Is that in the -200 to +1200 ms interval of an event?In Figures 3, 5, and 6, the downward notch at the end of most tracings seems to be an artefact of filtering the average ERP. If these notches are artefacts, rather that brain responses, this should be explained.The legend of Figure 4 gives the interval of interest as 600-1000 ms instead of 600-900 ms.On page 11 in the Dataset box, remove “32”", "responses": [ { "c_id": "1529", "date": "27 Aug 2015", "name": "J. Bruno Debruille", "role": "Author Response", "response": "The scientific study of consciousness is extremely difficult. Any serious attempt should therefore be regarded with a positively open mind. The intellectually challenging results of the present experiment, however, are unlikely to pertain to understanding the nature of qualia in the human brain. They rather seem to hint at a social cognition effect.We acknowledge the possibility that the differences found might not pertain to qualia. We now make this clear at several places. For instance in the discussion, last two sentences of last paragraph of page 12 of the pdf, which reads:“However, it is very important to point out that nothing in the present data can directly be related to qualia. The P600 effects of consistency only support spontaneous direct brain-to-brain influences that just could be related to consciousness.” We thus, nevertheless, believe, particularly after our new statistical analyses revealing an effect of consistency at each of the three electrode subsets, that the results point to direct brain-to-brain communications, in addition to social cognition effects, which are now studied and analyzed to address this issue. They appear different from the ERP effects of consistency. But, we do realize (and acknowledge in the paper) that the brain-to-brain communications might have nothing to do with consciousness. It has to be noted, however, that this possibility would then not be the most parsimonious, since it implies that qualia would have to be underlain by yet another phenomenon than the one responsible for the spontaneous brain-to-brain influences observed. DesignThe data consist in the average amplitude from 600 to 900 ms post stimulus onset of event related potentials (ERP) to complex stimuli selected as “most striking pictures” from the International Affective Picture System (IAPS). EEG recordings were obtained from dyads of socially related participants, simultaneously presented on separate halves of the same screen with a picture that could not be seen by the other participant, even though they were seated side by side and could see each other in the periphery of their visual field. Each participant had to observe the pictures in order to recognize them in a later memory test.Four different ERP conditions are compared, each consisting of the average of 70 stimuli (minus occasional artefacts at some channels) obtained from the same block. Two of the blocks were introduced to the two participants to consist of identical stimuli simultaneously presented to each of them, and the remaining two blocks were announced to consist of different sets of pictures. In each pair of conditions, this initial announcement was faithful once and contrary to the actual situation the other time. This allowed analyzing the situation in a 2 x 2 design of Sameness (Identical versus different pairs actually presented) by Belief (believing that the partner was presented with same versus different stimuli).The recordings, in each participant, were from 28 channels, referred to right ear lobe, not including any EOG dedicated channels. Each picture was presented for 1.0 s with a mean expected inter stimulus interval just below 1.15 s. Therefore, each of the four stimulus block lasted only about 2.5 minutes. The following simultaneous testing phase of the two participants required 28 minutes (280 targets + 280 new items each presented for 3.0 s).Although the rationale for the study considers qualia and the possibility of common brain signatures, the averaging over different stimuli in the same condition restricts considerably the relevance of the data to the original question of the nature of the qualia and of their physical support in the brain. The authors actually need to extend the concept of qualia to include the knowledge of whether the other person in the dyad is supposed to receive identical or different stimuli, which is very unlikely the dominant subjective experience (qualia) produced by any stimulus. The data are therefore much more likely to pertain to social cognition than to the intended exploration of a physiological substrate for the qualia. Actually, for features of visual perception that do not play a role in the task (for instance by being rare versus frequent events or relevant versus irrelevant), the ERP differences are likely to be idiosyncratic, requiring special statistical technique to detect differences that take different forms in different brains (e.g. Buchsbaum and Fedio, 1969, 1970, for the ERP difference between geometrical patterns and three letter words made of the same number of dots).ERP results.Visual inspection of the averages shows that the ERP were negative, relative to the 200 ms baseline, for the full duration of the stimuli in all four conditions for all 12 channels anterior to Cz, and were entirely positive in 6 of the seven posterior channels, at the level of, or posterior to Pz, excluding Pz itself which is mostly negative up to about 400 ms and positive thereafter in all four conditions. Despite the large anterior-posterior inversion in overall ERP polarity, the amplitude differences between conditions remain constant in polarity across all channels.The differences in condition are most marked between the two Believe Different conditions, with the Actually Same condition (SBd) being positive relative to the Actually Different condition (DBd). In the right hemisphere and posteriorly in the left hemisphere, the two Believe Same conditions (Actually Same and Actually Different, respectively SBs and DBs) were of intermediate amplitudes. In the frontal half of the left hemisphere, however, the ERP to the two Believe Same conditions overlap substantially with those of SBd, all three being less negative than DBd.The lack of inversion of the condition differences suggests that the underlying dominant effect is unlikely a simple modulation of the dominant sources of these ERP. The left frontal distinction from the remaining ordering of conditions could indicate the presence of at least two sources modulated by the experimental conditions.Although the data were analyzed with Belief and Sameness as factors, according to the experimental plan, it is relevant to decide whether the conditions SBs, SBd and DBs differ among themselves. Since the data are available, these could be tested. The following table gives the p value of all effects involving the three level Condition factor (DBd being excluded). effect Electrode group Sagittal Para sagittal Lateral Cond .774 .699 .863Cond x Hem .353 .228Cond x Elect .208 .128 .489Cond x Hem x Elect .066 .131 Thus, even without correcting for these 10 tests involving Condition, there is no indication of any difference in this group of three conditions. Since there was no condition in which the participants were alone, it cannot be decided empirically whether the effect of watching stimuli in dyads affects essentially the DBd condition or the remaining three conditions.The “operational hypothesis” that DBd would have a minimal impact on the ERPs implies that the topography in that condition is essentially that of the background activity. Since the background activity inverses polarity from front to back, this should also occur in the DBd condition. Since it does not, it becomes difficult to consider de facto DBd as the baseline no-effect condition. It could well be that DBd is the only condition that expresses the social cognition effect apparently present in the data.Although ad hoc, the following explanation may be proposed for the present data in terms of social cognition. In the DBd condition, each partner might be interested in whether the other person is presented with a similar amount of emotion. This would also be so at the beginning of the SBd condition, but the impression would rapidly build that the stimuli, although believed different, would be matched for emotion and therefore this interest in the amount of emotion felt by the other person could fade. This would account for the SDd-DBd difference being the most reliably detected. For the two Believe Same conditions, each partner would not doubt that the other person receives the same amount of emotion, resulting in no difference between these conditions. Many of the above comments end up casting doubts on the choice of the Different Believed-different (DBd) condition as a baseline condition. We acknowledge that this choice is in itself debatable. On the other hand, the differences obtained between this condition and the others were small (in the order of a microvolt) whereas standard deviations were in the order of 3.5 microvolts. Also, like the other conditions, DBd, was itself a bit noisy. Thus, it could have differed from the other conditions by chance. This could have happened because of the diversity of IAPS stimuli and because there was no speeded decision to make at each trial, which is known to produce sub-optimal ERPs.One way to deal with these problems was to further improve the signal to noise ratio by doubling the number of trials per condition and thus, by grouping conditions. This was doable, since after all, to test the fundamental hypothesis that there is some direct brain-to-brain communication that might have an effect on P600s, all that was needed was to see whether conditions that are actually consistent with the belief differed from the conditions where the stimulus displayed to the other participant was not consistent with belief. This is what we decided to do. Therefore, in each participant, we averaged together SBs with DBd (=conditions consistent with the belief) and DBs with SBd (=belief inconsistent conditions). The new grand averages are shown in the new Fig, 2. The ERPs look less noisy (still without any smoothing) and visual inspection suggests differences between consistency and inconsistency.Statistical analyses confirmed the significance of these ERP differences (see new results section). Because it was impossible for each participant to see and check whether the stimulus presented to his/her partner was consistent with the belief, which would have been the only classical means by which those differences could have been observed, then, we can say that these results support the existence of direct brain-to-brain communications.The possibility that the emotional reaction of the other participant to the stimulus could develop, be detected/processed by the subject and impact P600s was extremely unlikely. The time would be too short for the brain of the 1st participant to analyze the stimulus, to organize an appropriate emotional output to the muscles, then, for the muscles to actually move, for the other participant to detect these moves in his/her very peripheral visual-field, to process them and to detect their inconsistency with his own emotion in 600 ms. To further strengthen this timing argument, we used the small ERP differences that can be detected by the visual inspection of Fig, 2 in the N450 time window, especially at frontal electrode sites (e.g., Fz). We analyzed them. They were significant (now in the new results section), suggesting that the timing was even shorter (about 400 ms), further discarding the emotional account. These new results are now used in the discussion to support the existence of direct brain-to-brain communications. The part of the reviewer’s comment that we underlined is now addressed by computing the ERPs according to “whether the other person in the dyad is supposed to receive identical or different stimuli” New statistical analyses were run to test the significance of the small ERP differences found at left anterior electrode scalp site. They were significant, but the scalp distribution of these social cognition effects was quite different from that of the consistency effect (see below). CorrelationsThe result section also includes correlations calculated for P600 amplitude differences between conditions. These are reported, in Table 4, only for “the conditions that were the most different from each other, namely, SBd-DBd and DBs-SBs”. This justification seems incorrect, given the above table showing no detected difference between SBd, DBs and SBs, and since additional tests of the latter pair does not show any difference at any channel (all p>=.073). Reproducing the scatter plots of Figure 7 confirms that only 14 intact dyads were actually retained (an odd numbered participant followed by the next even number in the data base provided; the rationale for rejecting some participants should have been expressed). The situation, however calls for using the intra class correlation coefficient (ICC) in which no distinction is made as to which member should be A and which should be B, and for which only the common sample mean and common sample variance are used, resulting in one extra degree of freedom for the test, since only one mean is fitted to the data. When ICC are calculated, the following can be obtained (the sign after the channel name duplicates that of the correlation).ICC in SBd-DBd: 4 channels with p<.05 9 r=-0.5674 t(13)=-2.4843 p=0.0274* F8- 17 r=-0.5438 t(13)=-2.3363 p=0.0361* Fz- 20 r=-0.6196 t(13)=-2.8462 p=0.0138* P3- 22 r=-0.6207 t(13)=-2.8545 p=0.0135* Pz-ICC in ICC in DBs-DBd: no channel with p<.05ICC in ICC in SBs-DBd: no channel with p<.05ICC in SBd-SBs: 1 channel with p<.05 21 r= 0.6947 t(13)=3.4823 p=0.0040** P4+ICC in SBs-DBs: 3 channels with p<.05 18 r= 0.5142 t(13)= 2.1617 p=0.0499* O1+ 19 r= 0.5305 t(13)= 2.2562 p=0.0419* O2+ 21 r= 0.6208 t(13)= 2.8551 p=0.0135* P4+The other condition differences were not tested. Note that 8/140 (perhaps not independent) correlations tested have p<.05, while 7/140 is expected for independent statistical tests when H0 is true. The critical value of p, with a Bonferroni correction (for 140 independent tests) would be .0018, not reached by any of the 140 tests.Besides the possibility of there being no true correlation between the dyad members, the negative correlations between the dyad members, observed for the SBd-DBd difference, are counterintuitive. One could call upon disentanglement in which collapsing of the wave function for a particle causes the collapse of the complementary state, even at great distances. But here we have negative correlations on average amplitude differences. The quantum physics speculation not only would dispose of the phenomenon, assuming it is not a statistical accident, as being apparently explained, but this would require further ad hoc speculations to explain that the wave function would systematically collapse in the same way in the same person in the given test situation.If there is a true phenomenon to understand, we should start by questioning whether the negative correlations are mostly associated with SBd or with DBd. Note that since the same variable is used for both members of the dyads, changing its sign would not alter the direction of the correlation. It is unlikely that DBd is the source of the negative correlations since no significant (p<.05) correlation is seen in any other difference involving DBd. But the source of the negative correlation is not likely to be SBd either since the other tested difference involving SBd does not replicate the negative correlations. While the negative correlations for SBd-DBd were significant (p<.02) at P3 and Pz, it is a positive correlation that is significant (p<.01) at P4 for SBd-SBs. Since P4 also gives a significant (p<.02) positive correlation for SBs-DBs, the positive correlation for SBd-SBs is more likely attributable to SBs than to SBd (which was involved in the negative correlation).Setting aside the problem of ascribing the negative correlation to one of the involved experimental conditions, some important insight about social cognition could come from trying to identify the characteristics which defines which member of a dyad would produce a large or positive difference between conditions and which one would produce small or negative differences. Could this, for instance, characterize an implicit cognitive domination-submission attitude? But whether any of these correlations reflects a true correlation remains to be established first. The above discussion casts serious doubts on this. Response: We agree, and given, that these correlationswere not based on a priori hypothesis,rested on single rather than on the new grouped conditions and were not critical to discuss the possibility of a direct brain-to-brain communications, we decided to remove them. Thus, they are not in the new version of the paper.As to the comment:“The quantum physics speculation not only would dispose of the phenomenon, assuming it is not a statistical accident, as being apparently explained, but this would require further ad hoc speculations to explain that the wave function would systematically collapse in the same way in the same person in the given test situation”, We do not see why this systematicity would be mandatory for the system to detect inconsistency. It seems to us that this could happen in a particular way at each trial. Nevertheless, a real answer to this comment would involve physics far beyond our competences. For instance, what if being closely related to someone depends on some quantum entanglement, as audaciously proposed by some authors? We contacted Basil Hiley. He globally supported the paper but did not comment these issues. We tentatively imagine that the two entangled partners could act on quantum field energy in a similar way when seeing the same stimuli and knowing it and act on that field in different ways when seeing different stimuli while being told they are looking at the same. We know that anything that is produced by a biological organism tend to be feedback regulated. Accordingly, the brain could be sensitive to some changes in the quantum field energy. It might then also capture something of the effect of the entangled partner on that field. This could lead to inconsistency detection whatever the pattern of the change, provided that this pattern differs from the one expected. Could that be plausible? We would like to have expert opinions. We hope the paper will be of interests to physicists. However, many of them seem to adhere at Tegmark’s rebuttal of the link between consciousness and quantum field energy (as the one made by Penrose). They no longer discuss such link.Technical detailsIn reporting the behavioral data in Table 5 (now Table 3), the misses do not need to be reported, as they should be 70 minus the number of hits (not exactly so here probably because of rounding errors), but the false alarm rate for the 280 new pictures should be added to allow estimating the amount of guessing. Response: We believe we have to disagree. The amount of guessing for new stimuli is irrelevant since it cannot be used to differentiate the two belief conditions of the study phase. The aim of this memory test was not classical. It was to test the hypothesis of a different encoding in memory according to the conditions of the study phase. In Figure 2, it is not clear to what the 20 cm distance applies. It would be relevant, however to know how far apart from each other were the participants and at what distance from the screen were their eyes, so we can appreciate how much they could see of each other. Response: Thanks a lot for pointing to that figure. There were errors. The distance from the eyes to the screen was about 60 cm. The piece of cardboard separating the two halves of the screen was 41cm long. The distance between the heads of the 2 participants of a pair (i.e., from the right ear of the participant on the left to the left ear of the participant on the right) was at about 40 cm. We replaced Figure 1 by a photo on which these distances were added, as well as the visual angle corresponding to the width of the stimuli used. Instead of the first and last half of participants, Figures 5 et 6 could provide the means form the participants on the left and those on the right (even though they are not the same in number), so that any tendency to gaze at the other person at some systematic time after stimulus delivery would be reflected by opposite shifts at F7 and F8. From the data available, there is no systematic group difference if F8-F7 in any or the four conditions, but that tells nothing of the 0-600 ms interval. Great idea. We did it. Now Fig 5 A & B. And, I did not support the idea of opposed eye movement (included in the results section).If systematic correlations existed between dyad members, the independence of participants in the ANOVA would not be achieved, so that the degrees of freedom would actually be inflated. This possible bias could be acknowledged, even though the presence of correlations is not very convincing. We agree. We now mention that brain–to-brain communications jeopardize the independence of participants and that the degrees of freedom could actually be inflated. This is now in the discussion, p 12, 2nd paragraph, starting line 7. On page 6: the statement “electrodes for which amplitude exceeded +/- 100 mV were discarded” would need clarification about the period of time in which this would be observed, since event exclusion was done channel by channel. Is that in the -200 to +1200 ms interval of an event? Thanks for that too. There was a 1000-1200 ms non-reject interval. So trials were amplitudes outside of the+/-100 microvolts were within this late time window were kept. This is now mentioned (p6, last paragraph of 1st column, line 6).In Figures 3, 5, and 6, the downward notch at the end of most tracings seems to be an artefact of filtering the average ERP. If these notches are artefacts, rather that brain responses, this should be explained.The legend of Figure 4 gives the interval of interest as 600-1000 ms instead of 600-900 ms. This downward notch at the end of most tracings correspond to the off-effect (i.e., the disappearance of the IAPS picture used as a stimulus), which might also be associated to an eye movement. This is now mentioned in the legend of Fig 2 and 3. We do not think it could be an artifact of filtering. It was probably due to the 1000-1200 ms non-rejection time window. On page 11 in the Dataset box, remove “32” Done. Note that this data set now includes the tables for the social-cognition (belief) effect together with that for the consistency effect within the P600 time-window and that for this effect in the N400 time window." } ] } ]	1	https://f1000research.com/articles/3-316
https://f1000research.com/articles/7-288/v1	07 Mar 18	{ "type": "Case Report", "title": "Case Report: Pulmonary and Liver Sarcoidosis Suspected of Metastasis", "authors": [ "Behnam Jafari", "Gholamabas Sabz", "Elahe Masnavi", "Roghaye Panahi", "Saeid Jokar", "Amrollah Roozbehi", "Sajad Hasanzadeh", "Behnam Jafari", "Gholamabas Sabz", "Elahe Masnavi", "Roghaye Panahi", "Saeid Jokar", "Amrollah Roozbehi" ], "abstract": "Introduction: Sarcoidosis is a granulomatous disease with unknown cause that can vary from an asymptomatic condition. Almost half of the patients with sarcoidosis have no symptoms. In this article, we describe a sarcoidosis patient with lung and liver engagement; it may be confused with metastasis. Case report: A 39-year-old man, known as hypothyroidism who had come to the emergency ward with dyspnea and coughing after exposure to detergents in a closed environment A 39-year-old man, known as hypothyroidism who had come to the emergency ward with dyspnea and coughing after exposure to detergents in a closed environment. The patient smoked for 10 years (3 pack/year). No other findings were found in clinical examinations except for wheezing in the right lung. The patient's chest radiography was shown a mass. For further investigation, spiral CT scan was performed. Large lymph nodes on the right side of the trachea, measuring about 23 mm and a mass of 70 × 77 mm in the vicinity of the right lung hilum and a hypodense nodule in the posterior part of the liver with malignancy suspicious were reported. After several biopsy results was shown chronic granulomatous inflammation, the most important differential diagnosis is tuberculosis (TB) and sarcoidosis. Sputum smear, culture, and PCR were performed for tuberculosis. Also, the level of angiotensin-converting enzyme (ACE) was measured for sarcoidosis. the results ruled out TB and shown a higher level of ACE (ACE = 88).After diagnosis treatment started with prednisolone. Now, the patient is in the follow- up. Conclusion: In hilar lymphadenopathy of lung sarcoidosis is the importance differential diagnosis that should be considered", "keywords": [ "Sarcoidosis", "liver", "lung", "hilar lymphadenopathy" ], "content": "Introduction\n\nSarcoidosis is a granulomatous disease with unknown cause that can vary from an asymptomatic condition to being life-threatening1. Almost half of the patients with sarcoidosis have no symptoms. In some cases, with the help of chest radiography findings looking for other pathologies, it is diagnosed. Since the lungs are often involved, patients usually come to the clinic with lung complaints (such as shortness of breath, cough)2,3. Some of the clinical manifestations of sarcoidosis have a poor prognosis, including treatment-resistant lung sarcoidosis (pulmonary fibrosis, pulmonary hypertension), cardiac sarcoidosis, neurosarcoidosis, and multiple organ sarcoidosis. These clinical manifestations are often not diagnosed until the end stage of the disease, and their response to treatment is low. The incidence of severe sarcoidosis is the most common cause of death4. On time diagnosis, treatment and follow-up of patients reduces mortality. In this article, we describe a sarcoidosis patient with lung and liver involvement, which may be misdiagnosed as cancer metastasis.\n\n\nCase report\n\nA 39-year-old man, taxi driver, known to have hypothyroidism (being treated with levothyroxine) presented to the emergency ward with dyspnea and coughing after exposure to detergents in a closed environment. The patient was smoker (3 pack/year). The only in clinical examinations except for wheezing in the right side of chest. The patient's chest radiography identified a mass. For further investigation, a spiral computerized tomography (CT) scan was performed. Lymph nodes were enlarged on the right side of the trachea, measuring about 23 mm with a mass of 70 × 77 mm, in the vicinity of the right lung hilum. A hypodense nodule in the posterior part of the liver, suspected to be malignant, was also reported (Figure 1). The lesions were suspected to be metastatic tumors, therefore, a biopsy of the mass was performed via bronchoscopy. The biopsy results were reported as chronic inflammation and mucosal hyperplasia without malignancy, which did not conform to the CT report. The CT has repeated again, and confirmed the previous CT report. A CT guided mass biopsy was performed for pathological evaluation. The result showed chronic granulomatous inflammation, the two most likely causes being tuberculosis (TB) and sarcoidosis. Sputum smear, culture, and PCR were performed to test for TB, and angiotensin-converting enzyme (ACE) levels were measured for sarcoidosis. The results ruled out TB and showed high levels of ACE (ACE = 88 (normal 8-53)). Two months after the first visit, sarcoidosis was diagnosed and treatment started with prednisolone. Ophthalmology test for eye evaluation, echocardiography for cardiac evaluation and EMG/NCV (Electromyogram test and nerve conduction study) for evaluation of the nervous system were also performed to determine if there was any extra-pulmonary sarcoidosis, however, no lesions were found. After treatment by corticosteroid the symptoms of the patient subsided. Now the patient is on follow-up. Figure 2 show the CT scan of patient after treatment.\n\nCT scan in-patient with sarcoidosis-(A) Pulmonary lymphadenopathy and (B) granulomatous lesion in Liver involvement.\n\nCT scan of patient 6 months after treatment showing decreased size of pulmonary lymphadenopathy and (A) and improvement of hepatic lesions (B).\n\n\nDiscussion\n\nSarcoidosis occurs mainly in people aged 20 – 40 years and more common in females5. Diagnosis is based on medical history, granuloma manifestations in at least two different structures, staining and negative culture for acid-fast bacilli, a lack of occupational and internal exposure to toxins and absence of drug-related illnesses. It is difficult to determine the prevalence and incidence of sarcoidosis without clinical symptoms. In countries where Mycobacterium tuberculosis is common, sarcoidosis may not be detected5–7. The first evaluation of patients suspected of sarcoidosis include; cell blood count, serum biochemistry including creatinine, calcium, liver enzymes, alkaline phosphates, urine analysis, serum protein electrophoresis, inflammatory markers, lactate dehydrogenase, level of enzyme (ACE), and Complete Pulmonary Function Tests that should be performed in patients with respiratory symptoms or abnormalities of the lung parenchyma. In all cases, Mycobacterium and fungal disease should be considered, as it has a similar history (chronic cough) and clinical image (pulmonary lymphadenopathy)8. A few clinical case report has shown that sarcoidosis is less common in smokers9,10. Sarcoidosis has many clinical manifestations and affects all organs of the body. The lung is involved in at least 90% of sarcoidosis patients. Skin, eyes, liver and peripheral lymph nodes, with a frequency of 10 to 30% for other involved organs10. Cardiac involvement occurs in 25% of cases, but only causes clinical problems in 5% of cases, although it may suddenly be fatal, so it is important to examine all patients for cardiac sarcoidosis11,12. All patients should also be screened for eye involvement as it can cause visual impairment13. Sarcoidosis, like drugs, poisons, viral infections, and flukes induce liver dysfunction14,15. Up to 35% patients have abnormal liver function tests that are not related to the degree of disease16,17. Serum levels of ACE are increased in 60% of patients and have been shown to correlate with the level of disease activity. This test is non-invasive and is highly effective because the enzyme is produced by epithelial granuloma cells and its serum level reflecting the entire granulomatous activity in the body8,18. Fatigue was reported in over 50% of patients, which has a major effect on the quality of life. Pain was reported in 70% of patients. Arthralgia is the most common type of pain; a headache and chest pain also being reported.7,19. In terms of treatment, Corticosteroids are a selective therapeutic drug, and methotrexate and hydroxychloroquine are often alternative drugs20–22. Our patient had no abnormal blood counts, liver test dysfunction, and non-specific inflammatory markers abnormalities, only an increased titer of ACE with addition liver symptoms that included itching, jaundice, fever and abdominal pain.\n\n\nConclusion\n\nIn hilar lymphadenopathy of lung, sarcoidosis is an importance differential diagnosis that should be considered and prior to biopsy of lymph nodes and any invasive procedures, ACE enzyme levels should be measured.\n\n\nData availability\n\nNo data is associated with this article.\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 author(s) declared that no grants were involved in supporting this work\n\n\nReferences\n\nHunninghake GW, Costabel U, Ando M, et al.: ATS/ERS/WASOG statement on sarcoidosis. American Thoracic Society/European Respiratory Society/World Association of Sarcoidosis and other Granulomatous Disorders. Sarcoidosis Vasc Diffuse Lung Dis. 1999; 16(2): 149–73. PubMed Abstract\n\nLynch JP 3rd, Kazerooni EA, Gay SE: Pulmonary sarcoidosis. Clin Chest Med. 1997; 18(4): 755–785. PubMed Abstract \| Publisher Full Text\n\nLannuzzi MC, Rybicki BA, Teirstein AS: Sarcoidosis. N Engl J Med. 2007; 357(21): 2153–2165. PubMed Abstract \| Publisher Full Text\n\nSwigris JJ, Olson AL, Huie TJ, et al.: Sarcoidosis-related mortality in the United States from 1988 to 2007. Am J Respir Crit Care Med. 2011; 183(11): 1524–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNewman LS, Rose CS, Bresnitz EA, et al.: A case control etiologic study of sarcoidosis: environmental and occupational risk factors. Am J Respir Crit Care Med. 2004; 170(12): 1324–30. PubMed Abstract \| Publisher Full Text\n\nDouglas JG, Middleton WG, Gaddie J, et al.: Sarcoidosis: a disorder commoner in non-smokers? Thorax. 1986; 41(10): 787–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Kleijn WP, De Vries J, Lower EE, et al.: Fatigue in sarcoidosis: a systematic review. Curr Opin Pulm Med. 2009; 15(5): 499–506. PubMed Abstract \| Publisher Full Text\n\nLieberman J: Elevation of serum angiotensin-converting-enzyme (ACE) level in sarcoidosis. Am J Med. 1975; 59(3): 365–72. PubMed Abstract \| Publisher Full Text\n\nRybicki BA, Major M, Popovich J Jr, et al.: Racial differences in sarcoidosis incidence: A 5-year study in a health maintenance organization. Am J Epidemiol. 1997; 145(3): 234–41. PubMed Abstract \| Publisher Full Text\n\nChen ES, Moller DR: Etiology of sarcoidosis. Clin Chest Med. 2008; 29(3): 365–77, vii. PubMed Abstract \| Publisher Full Text\n\nSharma OP, Maheshwari A, Thaker K: Myocardial sarcoidosis. Chest. 1993; 103(1): 253–258. PubMed Abstract \| Publisher Full Text\n\nSilverman KJ, Hutchins GM, Bulkley BH: Cardiac sarcoid: a clinicopathologic study of 84 unselected patients with systemic sarcoidosis. Circulation. 1978; 58(6): 1204–1211. PubMed Abstract \| Publisher Full Text\n\nBaughman RP, Lower EE, Kaufman AH: Ocular sarcoidosis. Semin Respir Crit Care Med. 2010; 31(4): 452–462. PubMed Abstract \| Publisher Full Text\n\nGhadei R, Eilami O, Jahanbin S, et al.: A Case Report: Liver Abscess Caused by Fasciola hepatica in Yasuj. Armaghane danesh. 2017; 21(10): 1022–8. Reference Source\n\nKasper D, Fauci A, Hauser S, et al.: Harrison’s principles of internal medicine. 19e. USA2015. 2015; 2719–2726. Reference Source\n\nNolan JP, Klatskin G: The Fever of Sarcoidosis. Ann Intern Med. 1964; 61(3): 455–61. PubMed Abstract \| Publisher Full Text\n\nKennedy PT, Zakaria N, Modawi SB, et al.: Natural history of hepatic sarcoidosis and its response to treatment. Eur J Gastroenterol Hepatol. 2006; 18(7): 721–6. PubMed Abstract \| Publisher Full Text\n\nBajpayee L, Govender P, Berman J, et al.: The Diagnostic Value Of Serum Angiotensin Converting Enzyme In Sarcoidosis: A Systematic Review And Meta-Analysis. In B104. SARCOIDOSIS: CLINICAL STUDIES ON DIAGNOSIS, PROGNOSIS AND THERAPY. American Thoracic Society. 2017; 195: A4760. Reference Source\n\nHoitsma E, De Vries J, van Santen-Hoeufft M, et al.: Impact of pain in a Dutch sarcoidosis patient population. Sarcoidosis Vasc Diffuse Lung Dis. 2003; 20(1): 33–39. PubMed Abstract\n\nJudson MA: An approach to the treatment of pulmonary sarcoidosis with corticosteroids: the six phases of treatment. Chest. 1999; 115(4): 1158–1165. PubMed Abstract \| Publisher Full Text\n\nBaughman RP, Winget DB, Lower EE: Methotrexate is steroid sparing in acute sarcoidosis: results of a double blind, randomized trial. Sarcoidosis Vasc Diffuse Lung Dis. 2000; 17(1): 60–66. PubMed Abstract\n\nBaughman RP: Therapeutic options for sarcoidosis: new and old. Curr Opin Pulm Med. 2002; 8(5): 464–469. PubMed Abstract \| Publisher Full Text" }	[ { "id": "31753", "date": "19 Mar 2018", "name": "Seyed Masoom Masoompour", "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 case study presents a man with cough and dyspnea following exposure with irritant agent. His chest imaging showed a right hilar mass. Although this is not an unusual presentation of sarcoidosis but confirms that not all mass is malignant.\nMy comments:\nWhat do you mean by \"The incidence of severe sarcoidosis is the most common cause of death\"? As Swigris et al. had published, did you mean \"The underlying cause of death in most patients with sarcoidosis was the disease itself.\"\n\nWould you please clarify and rephrase this sentence: \"The only in clinical examinations except for wheezing in the right side of chest\"\n\nThe authors please add the bronchoscopic findings; was there any mass or narrowing there.\n\nPlease add the coronal view of chest CT; considering chest physical finding, wheeze, the reader would like to see if there is any narrowing in right main and/or intermediate bronchus.\n\nPlease add the unit of measurement of ACE level.\n\nPlease add the histologic slide of biopsy to the manuscript.\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? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "33755", "date": "15 May 2018", "name": "Edward S. Chen", "expertise": [ "Reviewer Expertise Immunology of sarcoidosis" ], "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 case of pulmonary sarcoidosis arising in a country where tuberculosis may be more common.\nThere is an error in the Abstract where a sentence is repeated \"A 39-year-old man, known as hypothyroidism who had come to the emergency ward with dyspnea and coughing after exposure to detergents in a closed environment A 39-year-old man, known as hypothyroidism who had come to the emergency ward with dyspnea and coughing after exposure to detergents in a closed environment.\"\nIn the abstract, the authors should revise the sentence \"A 39-year-old man, known as hypothyroidism...\" this probably should be revised to \"A 39-year-old man with known hypothyroidism...\"\nOn page 3, please revise the sentence \"The incidence of severe sarcoidosis is the most common cause of death.\"\nOn page 3, the authors should provide information from the bronchoscopy procedure report as to whether there was airway narrowing visualized during the bronchoscopy. This is of particular interest since the patient's main radiographic abnormality is enlarged thoracic lymph nodes without any notable lung infiltrates (stage I chest x-ray).\nOn Page 4, please consider revising \"It is difficult to determine the prevalence and incidence of sarcoidosis without clinical symptoms.\" Perhaps rephrase to clarify that \"It is difficult to determine the prevalence and incidence of sarcoidosis since the number of patients who have sarcoidosis but do not have symptoms is unknown.\"\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? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-288
https://f1000research.com/articles/7-688/v1	01 Jun 18	{ "type": "Research Article", "title": "COPD, metabolic syndrome, respiratory symptoms, and functional incapacity in smokers, ex-smokers, and never-smokers aged 40-59 in Almaty, Kazakhstan: a cross-sectional study", "authors": [ "Baurzhan Zhussupov", "Almaz Sharman", "Dana Sharman", "Almaz Sharman", "Dana Sharman" ], "abstract": "Background: No study has reported the relationship between smoking status with chronic obstructive pulmonary diseases (COPD) and metabolic syndrome (MetS) in Kazakhstan. The aim of this study was to assess the associations between health outcomes, including COPD, MetS, respiratory symptoms, and functional incapacity, with the cigarette smoking status. Methods: The cross-sectional study recruited 500 smokers, 200 ex-smokers, and 200 never-smokers aged 40-59 in Almaty, Kazakhstan. Questions assessed socio-demographic, clinical characteristics, and smoking behavior. Blood glucose and lipid profiles were determined after overnight fasting. COPD was defined according to the GOLD 2017 statement. Respiratory symptoms and functional incapacity were assessed by the COPD Assessment Test (CAT) and 6-min walk test (6MWT), respectively. Logistic regression models were used to assess the associations. Results: The prevalence of COPD among smokers, ex-smokers and never-smokers were 5.5%, 3.0% and 3.0%, respectively. Respiratory symptoms based on CAT were more prevalent among smokers (42.8%) as compared to ex-smokers (42.8% vs 17.0%; aOR 3.43, 95% CI 2.25–5.23) and never-smokers (42.8% vs 12.5%; aOR 5.44, 95% CI 3.42–8.65). Current smokers were more likely to walk less than 450 meters during 6MWT as compared to never-smokers (16.5% vs 5.0%; aOR 3.72, 95% CI 1.86–7.44). No significant association was found between the smoking status with COPD and MetS. Conclusions: Respiratory symptoms are common among the current smokers, even if most of them had preserved pulmonary function defined by spirometry.", "keywords": [ "COPD", "respiratory symptoms", "smoking", "6-min walk test" ], "content": "Introduction\n\nTobacco smoking is the greatest preventable cause of death and illness in the world that attributed to more than 7 million deaths in 20151. Smoking is associated with elevated risk of mortality from lung and other cancers, heart disease, stroke, chronic obstructive pulmonary disease (COPD) and other respiratory diseases, and other conditions2. The Global Adult Tobacco Survey (GATS) conducted in Kazakhstan in 2014 found that 19.1% of the study population were daily smokers (36.9% of males and 3.2% of females) (http://apps.who.int/tobacco/surveillance/survey/gats/kaz_countryreport_en.pdf?ua=1). COPD accounted for 3.2 million deaths worldwide in 20153 and is the fourth leading cause of death globally4. In Kazakhstan, an expected 1.4 million people might have COPD based on estimations from neighboring countries5.\n\nMetabolic syndrome (MetS), or the set of specific cardiovascular risk factors, is also identified as a significant public health challenge that increases risk of developing type 2 diabetes, cardiovascular disease and several cancers6,7. MetS is prevalent among patients with COPD8. It was shown that cardiovascular disease, hypertension and diabetes in patients with COPD are associated with higher hospitalization and mortality rates9.\n\nThe presence of respiratory symptoms prompts to recognize and diagnose respiratory disease including COPD10 and is often used to evaluate a severity of disease11.\n\nSmoking cessation has proven to have significant health benefits, including reduction in cardiovascular and COPD morbidity and mortality12–15, and decrease in outset of respiratory symptoms14. Some studies demonstrated that smoking is associated with metabolic syndrome and quitting smoking is recommended for the prevention and management of metabolic syndrome16,17.\n\nAssessment of the associations between several health outcomes, including COPD, respiratory symptoms, MetS and functional incapacity, and cigarette smoking status has not been done in Kazakhstan.\n\nThis study investigated these associations by comparing prevalence of these outcomes in smokers, ex-smokers, and never-smokers aged 40–59 in Almaty City, Kazakhstan.\n\n\nMethods\n\nThe design of the study was described in detail in the study protocol published earlier18.\n\nThe study included three groups of male and female residents of City of Almaty between the ages of 40 and 59 (inclusive), those who: 1) currently smoke cigarettes (current smokers); 2) quitted smoking between 1 and 5 years ago (ex-smokers); 3) have never smoked regularly (never-smokers)19. Smokers and ex-smokers were individuals with a minimum of 10 pack-year smoking history. Pack-years were calculated by taking the average number of cigarettes smoked per day divided by 20 and multiplied by the number of years smoked. The actual sample size was 900 including 500 smokers, 200 ex-smokers, and 200 never-smokers. The data was collected from September 2017 to April 2018.\n\nCOPD. COPD is defined according to the Global strategy for the diagnosis, management, and prevention of COPD (COPD Gold) as a post-bronchodilator ratio of forced expiratory volume in one second (FEV1) to forced vital capacity (FVC) less than 70% detecting by spirometry testing11.\n\nRespiratory symptoms. The COPD Assessment Test (CAT) is a quick and useful tool to weigh impact of COPD symptoms on health-related quality of life20. Participants with a CAT score of 10 and more are considered having more severe respiratory symptoms11.\n\nMetS. We used the International Diabetes Federation definition (https://www.idf.org/our-activities/advocacy-awareness/resources-and-tools/60:idfconsensus-worldwide-definitionof-the-metabolic-syndrome.html). Specifically, subjects were considered to have MetS if they had had central obesity (waist circumference (WC) > 94 cm in males and >80 cm in females for Euripides; >90 cm in males and >80 cm in females for Asians) plus two and more of the following criteria: (1) hypertriglyceridemia, ≥ 150 mg/dL; (2) reduced HDL cholesterol, < 40 mg/dL in males and < 50 mg/dL in females; (3) high blood pressure, ≥130/85 mm Hg; high fasting plasma glucose (FPG), ≥ 100 mg/dL.\n\nFunctional incapacity. The six-minute walk test (6MWT) determines functional exercise capacity in patients with moderate-to-severe heart or lung disease21. There are several reference systems predicting distance of 6MWT in healthy subjects22. They take into account subject’s gender, age, height and weight. However, no study has been conducted so far to develop such a system for the Kazakh population. Therefore, we consider the distance of 450 meters as a cut-off level to define functional incapacity because distances less than this value is highly correlated with maximal oxygen capacity23.\n\nData were analyzed using R, a language and environment for statistical computing, version 3.3.1 (https://www.R-project.org/). Logistic regression analyses were utilized to measure the crude and adjusted associations between the smoking status and the four health outcomes. Crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. For adjustment, logistic regression models included gender (categorical), age (continuous), ethnicity (categorical), level of education (categorical), secondary smoking (for the ‘ex-smokers vs never-smokers’ comparison, categorical), pack-year (for the ‘smokers vs ex-smokers’ comparison, continuous). An alpha-level of 0.05 was chosen to test statistical significance. The Bonferroni Correction of the significance level was applied to account that three associations (smokers vs ex-smokers, smokers vs never-smokers, and ex-smokers vs never-smokers) were studied for each health outcome. Therefore, a P-value<0.017 (0.05/3) was considered significant.\n\n\nResults\n\nBasic characteristics and smoking patterns of study participants by study group are summarized in Table 1. The study participants were approximately equally distributed as those who were younger than 50 years and older than 50 years of age. There were about twice as many participants of Kazakh ethnicity (Asian origin) compared to Russians (European origin). Most of the participants had higher education. Majority of those who were smoking had more than 20 pack-years of smoking (61%).\n\n* The number of cases (percentage).\n\nData on COPD prevalence, CAT-test, functional capacity based on 6MWT and prevalence of MetS are shown in Table 2. The prevalence of COPD among smokers, ex-smokers and never-smokers were 5.5%, 3.0% and 3.0%, respectively. Respiratory symptoms based on CAT were more prevalent among smokers (42.8%) as compared to ex-smokers (17.0%) and never-smokers (12.5%). Proportion of participants with MetS varied across the study groups from 28.6% among never-smokers to 37.5% among currents smokers. Current smokers were more likely (16.7%) to walk less than 450 meters during the 6MWT as compared to never-smokers (5.0%).\n\nNotes. COPD = chronic obstructive pulmonary disease; CAT = COPD Assessment Test; MetS = metabolic syndrome; SD=standard deviation; 6MWT=6-minite walk test.\n\n* The number of cases (percentage).\n\nMean scores of CAT components are show in Figure 1. Smokers had significantly worse mean scores in all CAT components than ex-smokers and never-smokers except in the sleeping category. There were statistically significant (p<0.017) differences between mean scores of ex-smokers and never-smokers for cough, phlegm, and chest tightness.\n\nThe relationship between the smoking status and the prevalence of metabolic syndrome components is presented in Table 3. The prevalence of reduced levels of high-density lipoproteins was significantly higher for the group of current smokers than for ex-smokers and never smokers. No significant relationship was found between the prevalence of other components and the smoking status (P>0.017).\n\nNotes. MetS = metabolic syndrome; TG= triglyceride; HDL= high-density lipoprotein; FPG= fasting plasma glucose; BP=blood pressure.\n\np<0.017 for each pairwise comparison (SE – smokers vs ex-smokers; SN – smokers vs never-smokers; EN – ex-smokers vs never-smokers) by the chi-square test.\n\n† The number of cases (percentage).\n\nCrude and adjusted odds ratios (aOR) between COPD, respiratory symptoms, MetS, functional incapacity and the smoking status are presented in Table 4. Current smokers were significantly associated with increased prevalence of functional incapacity (6MWT<450 meters) while comparing with never-smokers (aOR 3.72, 95% CI 1.86–7.44). Current smokers had more prevalent respiratory symptoms than ex-smokers (aOR 3.43, 95% CI 2.25–5.23) and never-smokers (aOR 5.44, 95% CI 3.42–8.65). No significant association was found between the smoking status and prevalence of COPD and MetS in all three pairwise comparisons.\n\nNotes. COPD = chronic obstructive pulmonary disease; CAT = COPD Assessment Test; MetS = metabolic syndrome; 6MWT=6-minite walk test; OR=odds ratio.\n\np<0.017\n\n\nDiscussion\n\nThis study was the first in Kazakhstan to assess prevalence of COPD, respiratory symptoms, MetS, functional exercise incapacity in adults aged 40–59, those who smoked cigarettes, quit smoking and never smoked. The study demonstrated that at least 3% of the study population were affected by COPD. This value is much higher than the rate of previously reported COPD cases of 340.5 per 100,000 population (http://www.mz.gov.kz/en/pages/statistical-symposium-health-republic-kazakhstans-population-and-activity-health-service).\n\nTwo of four health outcomes assessed in the study, respiratory symptoms and functional exercise incapacity, were significantly worse among current smokers than among those participants who never smoked cigarettes. Two other study outcomes, COPD and MetS, were more prevalent in current smokers, but the study couldn’t demonstrate statistically significant association with the smoking status.\n\nThe presence of all symptoms and signs of respiratory disease that are used as CAT components were significantly lower among ex-smokers compared to those who continued smoking. This study is in line with other studies, suggesting that respiratory symptoms improve after smoking cessation, however remain worse or at least similar to those found in never-smokers19. One may assume that respiratory symptoms may be a significant reason for smoking cessation in a number of cases.\n\nIt is important to notice that a substantial proportion of the current smokers or 42.8% had respiratory symptoms, even if most of them had preserved pulmonary function defined by spirometry. It was shown that high CAT scores in current or ex-smokers are associated with exacerbation-like events, i.e. that symptomatic smokers with preserved pulmonary function have evidence of airway disease even when they do not meet the current definition for COPD24. Exacerbation-like events in individuals without COPD raise the burden of respiratory disease and are associated with considerable health outcomes25. Thus, even proper diagnosis of COPD based on the spirometric criterion substantially underestimates the effect of smoking on the individual with smoking history.\n\nThe study has several limitations. This was cross-sectional study that was not designed to determine causal relationship. Second, although we adjusted for the known potential confounders, residual bias attributable to uncontrolled confounding variables may exist and cannot be estimated. Third, we used a quota sample which may have affected the generalizability of the study results to the study population. Fourth, our study participants were adults aged 40–59 from Almaty city, and further assessment is needed to determine whether the results are applicable to the other population groups.\n\n\nData availability\n\nDataset 1. Raw data behind the results of this study.\n\nThe main data are provided for 900 subjects who completed the study (500 smokers, 200 ex-smokers, and 200 never-smokers). Missing data were not replaced.\n\nSubject ID (ID), study group, gender, age, ethnicity, education, cigarette consumption (expressed as a number of pack-year), regular secondary smoking in past 12 months (for ex- and never-smokers), airflow obstruction by spirometry (FEV1/FVC < 70%), Six minute walk test, CAT total score, CAT components: - cough; - phlegm; - tightness of chest; - shortness of breath (SOB); - limited doing activities at home; - not confident leaving home; - not sleeping soundly; - no energy, metabolic syndrome (1 – yes, 0 – no), central obesity (1 – yes, 0 – no), raised triglyceride (≥ 150 mg/dL: 1 – yes, 0 – no), reduced high-density lipoprotein (< 40 mg/dL for males and < 50 mg/dL for females: 1 – yes, 0 – no), raised blood pressure (≥130/85 mm Hg: 1 – yes, 0 – no), raised fasting plasma glucose (≥ 100 mg/dL: 1 – yes, 0 – no). F1000Research: Dataset 1. Raw data behind the results of this study, 10.5256/f1000research.14614.d20345626\n\n\nEthics and consent\n\nThe National Central Ethic Committee under Ministry of Health and Social Development of the Republic of Kazakhstan approved this study on August 19, 2016. Written informed consent was obtained from all participants. The study has been registered in ClinicalTrial.gov (the release date: October 3, 2016; Identification No. NCT02926534)27.", "appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe project is partially funded by a grant from Philip Morris International (IIS.PMI.2016.001).\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\nThis study is supported with resources and the use of facilities at Kazakhstan Academy оf Preventive Medicine, the HealthCity Clinic and Synergy Group Kazakhstan.\n\n\nReferences\n\nGBD 2015 Risk Factors Collaborators: Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016; 388(10053): 1659–1724. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Global Health Risks: Mortality and Burden of Disease Attributable to Selected Major Risks. Geneva: WHO; 2009. Reference Source\n\nGBD 2015 Chronic Respiratory Disease Collaborators: Global, regional, and national deaths, prevalence, disability-adjusted life years, and years lived with disability for chronic obstructive pulmonary disease and asthma, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet Respir Med. 2017; 5(9): 691–706. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLozano R, Naghavi M, Foreman K, et al.: Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012; 380(9859): 2095–128. PubMed Abstract \| Publisher Full Text\n\nAdeloye D, Chua S, Lee C, et al.: Global and regional estimates of COPD prevalence: Systematic review and meta-analysis. J Glob Health. 2015; 5(2): 020415. PubMed Abstract \| Free Full Text\n\nReaven P: Metabolic syndrome. J Insur Med. 2004; 36(2): 132–42. PubMed Abstract\n\nRusso A, Autelitano M, Bisanti L: Metabolic syndrome and cancer risk. Eur J Cancer. 2008; 44(2): 293–7. PubMed Abstract \| Publisher Full Text\n\nCebron Lipovec N, Beijers RJ, van den Borst B, et al.: The Prevalence of Metabolic Syndrome In Chronic Obstructive Pulmonary Disease: A Systematic Review. COPD. 2016; 13(3): 399–406. PubMed Abstract \| Publisher Full Text\n\nMannino DM, Thorn D, Swensen A, et al.: Prevalence and outcomes of diabetes, hypertension and cardiovascular disease in COPD. Eur Respir J. 2008; 32(4): 962–9. PubMed Abstract \| Publisher Full Text\n\nHayward RA, Chen Y, Croft P, et al.: Presentation of respiratory symptoms prior to diagnosis in general practice: a case-control study examining free text and morbidity codes. BMJ Open. 2015; 5(6): e007355. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVogelmeier CF, Criner GJ, Martinez FJ, et al.: Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Lung Disease 2017 Report. GOLD Executive Summary. Am J Respir Crit Care Med. 2017; 195(5): 557–582. PubMed Abstract \| Publisher Full Text\n\nCarreras G, Pistelli F, Falcone F, et al.: Reduction of risk of dying from tobacco-related diseases after quitting smoking in Italy. Tumori. 2015; 101(6): 657–63. PubMed Abstract \| Publisher Full Text\n\nMons U, Müezzinler A, Gellert C, et al.: Impact of smoking and smoking cessation on cardiovascular events and mortality among older adults: meta-analysis of individual participant data from prospective cohort studies of the CHANCES consortium. BMJ. 2015; 350: h1551. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu Y, Pleasants RA, Croft JB, et al.: Smoking duration, respiratory symptoms, and COPD in adults aged ≥45 years with a smoking history. Int J Chron Obstruct Pulmon Dis. 2015; 10(1): 1409–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTønnesen P: Smoking cessation and COPD. Eur Respir Rev. 2013; 22(127): 37–43. PubMed Abstract \| Publisher Full Text\n\nYu M, Xu CX, Zhu HH, et al.: Associations of cigarette smoking and alcohol consumption with metabolic syndrome in a male Chinese population: a cross-sectional study. J Epidemiol. 2014; 24(5): 361–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPérez-Martínez P, Mikhailidis DP, Athyros VG, et al.: Lifestyle recommendations for the prevention and management of metabolic syndrome: an international panel recommendation. Nutr Rev. 2017; 75(5): 307–326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharman A, Zhussupov B, Sharman D, et al.: Cross-Sectional Study of Chronic Obstructive Pulmonary Disease Prevalence Among Smokers, Ex-Smokers, and Never-Smokers in Almaty, Kazakhstan: Study Protocol. JMIR Res Protoc. 2017; 6(7): e143. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWillemse BW, Postma DS, Timens W, et al.: The impact of smoking cessation on respiratory symptoms, lung function, airway hyperresponsiveness and inflammation. Eur Respir J. 2004; 23(3): 464–76. PubMed Abstract \| Publisher Full Text\n\nJones PW, Harding G, Berry P, et al.: Development and first validation of the COPD Assessment Test. Eur Respir J. 2009; 34(3): 648–54. PubMed Abstract \| Publisher Full Text\n\nATS Committee on Proficiency Standards for Clinical Pulmonary Function Laboratories: ATS statement: guidelines for the six-minute walk test. Am J Respir Crit Care Med. 2002; 166(1): 111–7. PubMed Abstract \| Publisher Full Text\n\nCasanova C, Celli BR, Barria P, et al.: The 6-min walk distance in healthy subjects: reference standards from seven countries. Eur Respir J. 2011; 37(1): 150–6. PubMed Abstract \| Publisher Full Text\n\nMorales FJ, Martínez A, Méndez M, et al.: A shuttle walk test for assessment of functional capacity in chronic heart failure. Am Heart J. 1999; 138(2 Pt 1): 291–8. PubMed Abstract \| Publisher Full Text\n\nWoodruff PG, Couper D, Han MK: Symptoms in Smokers with Preserved Pulmonary Function. N Engl J Med. 2016; 375(9): 895–7. PubMed Abstract \| Publisher Full Text\n\nTan WC, Bourbeau J, Hernandez P, et al.: Exacerbation-like respiratory symptoms in individuals without chronic obstructive pulmonary disease: results from a population-based study. Thorax. 2014; 69(8): 709–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhussupov B, Sharman A, Sharman D: Dataset 1 in: COPD, metabolic syndrome, respiratory symptoms, and functional incapacity in smokers, ex-smokers, and never-smokers aged 40-59 in Almaty, Kazakhstan. F1000Research. 2018. Data Source\n\nKazakhstan Academy of Preventive Medicine: Cross-Sectional Study of COPD Prevalence Among Smokers, Ex-smokers and Never-Smokers in Almaty, Kazakhstan. In: ClinicalTrials.gov [cited 2018 Apr 30]. Reference Source" }	[ { "id": "39276", "date": "22 Oct 2018", "name": "Alexandru Corlateanu", "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 aim of this study is not quite clear, yet the topic is very interesting. The abstract is irrelevant and incomplete, and the conclusion is not fully corresponding to the aim of the study. Citations are correctly referenced at the end, but please check for the two internet links that were mentioned in our specific comments. Appropriate key studies were included. It is clear what is already known about the topic, but it is not clear what research question is outlined. The relationship between COPD and metabolic syndrome needs more justification in the introduction. Process of the subject selection is clear, and the method is valid. Please add more details regarding the software used. Tables and Figures are clearly presented. Please add statistical significance for Tables 1, 2 and 3. Titles, columns, and rows are labelled correctly. Still not quite sure about what is statistically significant and practically meaningful. Results may be over-interpreted. The discussion is very uncertain and repetitive. Limitations are not fatal and there should be another opportunity to inform future research. Discussion section is low quality. The article is not consistent within itself:\n\n- Conclusions of the study reflecting only one aspect of the aim, and it is only respiratory symptoms.\n\n- There is no data presented on spirometry of patients from 3 groups. It is not clear if it was performed in all groups.\n\n- In the protocol of this study published earlier, it was mentioned that there was laboratory testing of serum: its not clear if alfa1antitripsin was tested.\n\n- The same for chest computed tomography: any data from the smoking group?\n\n- There is surprisingly low prevalence of COPD in the smoker group!\n\n- In the discussion, the data from the current study must be compared with the data from other studies.\nConclusions are not corresponding to the aim of the study.\nQuestions to the author(s): 1.\n\nHow reliable is the COPD Assessment Test (CAT) for this study? 2.\n\nWhat was the cause for COPD among never-smoker subjects? 3.\n\nIn your introduction, first paragraph: You used 2015 statistic numbers. Why not 2017 or 2018? 4.\n\nWhy is metabolic syndrome associated with COPD? Please consider adding a paragraph regarding the pathophysiology between those entities. 5.\n\nWhat impact you did consider having with this paper? 6.\n\nRegarding the statistical computing “R”: Where was it designed? Is it reliable and accurate?\n7.\n\nWhat was the etiology for the metabolic syndrome of those patients? 8.\n\nIs it possible that patients with metabolic syndrome had respiratory symptoms due to their obesity?\n\nSpecific comments from reviewer to author(s): 1.\n\nIntroduction, 3rd paragraph: Please use the correct internet citation for this paragraph (Vancouver style if possible) and place it as #3. Then move all the numbers according to their new assigned number citation. (for example: …”20153” now is “20154”.) 2.\n\nOutcome measures: MetS – Correct internet citation for the definition and also assigned a number as well. Then correct the reference list and check that all the numbers are according to their citation in the article. 3.\n\nPlease add more information about the statistical computing “R”.\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? 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?\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/7-688
https://f1000research.com/articles/7-32/v1	09 Jan 18	{ "type": "Method Article", "title": "META-pipe Authorization service", "authors": [ "Inge Alexander Raknes", "Lars Ailo Bongo", "Inge Alexander Raknes" ], "abstract": "We describe the design, implementation, and use of the META-pipe Authorization service. META-pipe is a complete workflow for the analysis of marine metagenomics data. We will provide META-pipe as a web based data analysis service for ELIXIR users. We have integrated our Authorization service with the ELIXIR Authorization and Authentication Infrastructure (AAI) that allows single sign-on to services across the ELIXIR infrastructure. We use the Authorization service to authorize access to data on the META-pipe storage system and jobs in the META-pipe job queue. Our Authorization server was among the first services that integrated with ELIXIR AAI. The code is open source at: https://gitlab.com/uit-sfb/AuthService2.", "keywords": [ "ELIXIR AAI", "SAML", "OAuth 2.0", "Authorization", "Authentication" ], "content": "Introduction\n\nELIXIR brings together and coordinates European life science resources, including databases, software tools, training materials, cloud storage, and supercomputers. One of the resources developed in the ELIXIR-EXCELERATE project is META-pipe1, an automated pipeline for annotation and analysis of metagenomic and genomic sequence data is targeted for marine metagenomics. We will provide META-pipe as a web based data analysis service for ELIXIR users.\n\nThe ELIXIR Compute Platform builds distributed cloud, compute, storage, and access services for the life-science research community. An important part of the cloud platform is the geographically distributed Authentication & Authorization Infrastructure (AAI) that provides services for identification, authentication, and authorization of ELIXIR end users. We have integrated our META-pipe Authorization service with the ELIXIR AAI, such that our users can use the single sign-on used across all ELIXIR services. Most users can sign-on using their home institution credentials; the remaining users can use Google, LinkedIn, or ORCID credentials. Our Authorization server was among the first services that integrated with ELIXIR AAI.\n\nIn this paper, we describe the design, implementation, and use of the META-pipe Authorization service. It limits which services and users are authorized to access and modify META-pipe datasets and job results. It authenticates users using ELIXIR AAI, and it authorizes access to data on the META-pipe storage system and jobs in the META-pipe job queue. We therefore use it as ad-hoc authentication for our own services.\n\nWe designed the META-pipe authorization service with three main design goals:\n\n1. Keep it simple and stupid; especially for creating new services.\n\n2. Separation of concerns. Keep authentication and authorization separate from the other META-pipe backend services.\n\n3. Stable interface to avoid doing the same work multiple times. We therefore use existing standards in the interaction between services as these are assumed to be reasonably defined and stable.\n\nOur solution is an external application decoupled from the rest of our services. It contains all the integration code needed to integrate with an external authentication service such as ELIXIR AAI, and it can be further be improved independent of the other META-pipe services. We also wanted it to be simple enough such that our small development team could implement it from scratch. We follow standards so we can re-use existing libraries and use proven, stable interfaces.\n\nExisting out-of-the-box solutions do not fulfill our requirements. For example, OpenID Connect is a large standard with many mandatory parts that we do not need for our service. We have therefore chosen standards, libraries, and tools that are simple and developer friendly, and that can easily be incorporated in any framework/programming language combination as our services use Scala and Go, and Java.\n\nThe rest of this paper describes the design and implementation of the server, how it is used by end-users and our backend services, and it gives an overview of the standards and libraries we have used. Finally, we outline ongoing and future work.\n\n\nMethods\n\nThis section summarizes the implementation and usage of the META-pipe authorization service. Additional details are in The META-pipe Authorization service design document.\n\nThe authorization service fits into the context of the META-pipe backend that has two Clients: the web application and the Galaxy tool. In addition, the context has several services, including storage and job management services. We have integrated our Authorization server with the ELIXIR AAI, which provides user identities from the ELIXIR federated IDP for European educational and research institutions, Google, LinkedIn, and ORCID. Users therefore use the single sign on ELIXIR web interface, and we rely on ELIXIR AAI to maintain (the federated) user databases.\n\nSince some external IDPs, such as the Norwegian Feide that is one of the federated ELIXIR AAI IDPs, requires single sign-out we needed a way to revoke tokens on a short notice. This requires a central service for validating tokens and is one of the reasons we chose to use Token Introspection over JWT Bearer Tokens (RFC-7523).\n\nThe authorization server implements the Authorization Code Grant in the OAuth 2.0 Authorization Framework (RFC-6749) (Figure 1). The server uses OAuth 2.0 Bearer Tokens (RFC-6750) as the access tokens since the Bearer Tokens are well supported by software libraries and they are required by the Token Introspection specification. In addition, the server implements an OAuth 2.0 Token Introspection (RFC-7662) endpoint that the Resource Servers can use to introspect the tokens, whereby getting information about what can be accessed and if the token is still valid.\n\nThe Authorization service is integrated with ELIXIR AAI. It is implemented to isolate the integration to the authorization server. We expose a REST API for the clients. The clients do not know about ELIXIR AAI.\n\nOur Meta-pipe web application stores the Access Tokens and Refresh Tokens in the browser's local storage.\n\nIn OAuth 2.0 the Access Token Scope is defined by the Authorization server. Since we use a REST architecture for our services, each resource has its own URI. We have defined the Access Token Scope such that a Resource Server can determine if a request is authorized by comparing the Access Token Scope with the requested URI and method.\n\nWe have integrated the META-pipe Authorization service with ELIXIR AAI. We use the OAuth 2.0 Authorization Code Grant since it is flexible enough to isolate the integration to the authorization server. We can therefore implement our Clients without them having to know neither about SAML nor ELIXIR AAI. We cannot use standards such as RFC-7522 (Security Assertion Markup Language (SAML) 2.0 Profile for OAuth2.0 Client Authentication and Authorization Grants) and the more general RFC-7521 (Assertion Framework for OAuth 2.0 Client Authentication and Authorization Grants), since these require the Client to directly interact with the Issuer and hence both the Client and the Authorization server need to know SAML and how to interact with the ELIXIR AAI. Another benefit of the Authorization Code Grant is that it has very good software library support.\n\nA client using the OAuth 2.0 Authorization Code Grant redirects the User Agent to the Authorization Endpoint and then, after the Resource Owner has authorized the request, the User Agent is redirected to a URI defined by the Client where an Authorization Code is included in the URI query parameter. The Client can then exchange this Authorization Code for an Access Token and a Refresh Token by querying the Token Endpoint.\n\nWhile RFC-6749 specifies how the Client interacts with the Authorization Server, it does not specify how the Authorization Endpoint obtains an authorization from the Resource Owner; this is left to the implementer of the Authorization Endpoint. Therefore, once the User Agent has been redirected to the Authorization Endpoint we can perform the authentication process that is required by the ELIXIR AAI (including any Use Agent interactions/redirects) if we are able to redirect the User Agent to the URI specified by the Client after the user has been authenticated.\n\nELIXIR-Norway uses Galaxy as a common GUI for the analysis services provided for Norwegian users: https://nels.bioinfo.no/. The Galaxy users are authenticated using the Feide authentication infrastructure (Feide technical guide, Feide integration guide) that is used by all Norwegian universities. We have integrated our ELIXIR-Norway Galaxy using an Apache HTTPD reverse proxy with AuthMemcookie and SimpleSAMLPHP. The Apache HTTPD proxies authenticated requests to the Galaxy web application with an HTTP header used to specify who is logged in.\n\nA Galaxy tool is defined by an XML file that describes how to launch a process and how the process' arguments should be presented in the Galaxy GUI. The tool developer maps every relevant parameter to a command line that Galaxy executes when the user runs the tool. The parameters include parameters selected by the user in the web GUI as well as a few contextual parameters, like the user's email address that is retrieved from Feide when the user authenticates.\n\nThe Authorization Server must know about ELIXIR AAI, the SAML protocol and the AAI specific attributes that provides information about a user. It must also have knowledge about which users are authorized to use which resources.\n\nThe Client must support OAuth 2.0 (RFC-6749) and Bearer Token Usage (RFC-6750).\n\nThe Resource server must support Bearer Token Usage (RFC-6750) and OAuth 2.0 Token Introspection (RFC-7662). It must also know how to interpret a Scope in the correct context of the application.\n\nWe have implemented the authorization service in the Dropwizard web framework. Dropwizard is a lightweight Java web framework aimed at creating micro services. It uses Apache Oltu for handling the OAuth protocol, and it stores all its state in a PostgreSQL database using Hibernate ORM. Apache Oltu is a library for implementing OAuth 2.0 servers in Java. It manages the OAuth 2.0 protocol and helps serializing responses and parsing/validating requests. Hibernate is an Object Relational Mapper.\n\nWe have implemented the META-pipe web application (Client) in JavaScript using the Client OAuth 2.0 library for interfacing with the authorization server and the Resource Servers. Client Oauth 2 (mulesoft/js-client-oauth2) is a JavaScript library for obtaining an authorization from an OAuth 2.0 Authorization server and for making authorized requests to a Resource Server. Client OAuth 2 supports several Authorization Grants including the Resource Owner Password Credentials Grant and Authorization Code Grant. It also supports Bearer Token usage (RFC-6750). Client OAuth 2 can be used from a web browser or from within NodeJS.\n\nOAuth 2.0 Token Introspection is not yet widely supported by software libraries, so we had to implement this from scratch. The specification is only 17 pages and it is very easy to implement. Our library implementation for Resource Servers is therefore only 176 lines of Go code.\n\nWe here list the standards that we used and that are useful for others that are developing a similar service. These contain key definitions, abstraction, and representations:\n\nThe OAuth 2.0 Authorization Framework (RFC-6749) standard for performing authorization describes the Acces Token, which is a key abstraction in OAuth 2 that provides an abstraction layer, replacing different authorization constructs (such as username and password) with a single token understood by the resource server.\n\nThe The Oauth2.0 Authorization Framework: Bearer Token Usage (RFC-6750) defines the Bearer Token that is passed directly to the Resource Server when performing an authorized request.\n\nThe JSON Web token (JWT) (RFC-7519) standard describes a compact, URL-safe means of representing claims transferred between two parties.\n\nThe OAuth 2.0 Token Introspection (RFC-7662) defines a method for inspecting a token. The Security Assertion Markup Language (SAML) 2.0 Profile for OAuth 2.0 Client Authentication and Authorization Grants (RFC-7522) describes how to use SAML to request an OAuth 2.0 access token and how to use SAML for client authentication.\n\n\nUse Cases\n\nHere we describe the request flow for three use cases: META-pipe web application, command line, and Galaxy. In addition, we describe the request flow for the REST API used to submit jobs for these three uses cases, and we discuss limitation for downloading of META-pipe results using the browser.\n\nTo authorize an end-user to run META-pipe analyses:\n\n1. The end-user (Resource Owner) clicks “Log in with ELIXIR AAI” in the META-pipe web application.\n\n2. The end-user is redirected to the Authorization Endpoint as defined by the Authorization Code Grant and then to the ELIXIR AAI using SAML web SSO.\n\n3. The end-user logs in via one of the IDPs that are supported by the ELIXIR AAI and is then redirected back to the web application via the authorization server where it obtains an Authorization Code.\n\n4. The web application obtains an Access Token by contacting the Token Endpoint with the Authorization Code.\n\nWe provide a command line tool to a few power users so they can submit multiple jobs simultaneously. The tool obtains an Access Token by using the Client Credentials Grant.\n\nWhen the META-pipe Galaxy tool (Client) is called by Galaxy the end user will already be authenticated using the Galaxy/Feide integration at https://galaxy-uit.bioinfo.no/.\n\n1. The Galaxy tool will get the user's email address as a command line parameter.\n\n2. The Galaxy tool will be trusted to access all users’ data and will obtain an Access Token and a Refresh Token using the Client Credentials Grant (RFC-6749) with a Scope that is limited to the requesting user's resources that are necessary to run a job.\n\n3. The Galaxy Tool will hold the Access Token and Refresh Token in memory for subsequent API requests until the job has completed, and the tool terminates. No authorization state will be persisted between multiple tool invocations.\n\nThe above three use-cases use the META-pipe REST API to either submit jobs or monitor their progress. When a Client contacts an API endpoint (Resource Server) the following happens:\n\n1. The Client provides the Bearer Access Token in the Authorization header to the API endpoint (RFC-6750).\n\n2. When the Resource Server receives the request, it will query the Introspection Endpoint to get the Scope of the Access Token and verify that it is still valid.\n\n3. If the Access Token is valid the Resource Server will compare the Access Token' Scope (as returned by the Introspection Endpoint) to the scope required to process the request. If the request is authorized it will process the request, otherwise it will respond with an appropriate error code (RFC-6750).\n\n\nSummary\n\nWe have described the design, implementation, and use of the META-pipe Authorization service. We use the Authorization service to authorizes access to data on the META-pipe storage system and jobs in the META-pipe job queue. Our Authorization server was among the first services that integrated with ELIXIR AAI.\n\nWhen downloading a data set (META-pipe result) from our storage service via the browser it is necessary to provide a direct link to the downloadable content while at the same time provide an Access Token to access the data. RFC-6750 allows for the use of an \"access_token\" URI query parameter containing a Bearer Token for authorization. A potential issue with this approach is that if a user shares a download link with other users they will get the user's access token. A solution to this is to limit the access token's Scope to only have read access to that particular resource, thus mitigating the risk of users inadvertently giving access to their account.\n\nWe plan to add an admin group for META-pipe administrators to a configuration file in the Authorization server. Another solution is to create a group in an Virtual Organization provided by ELIXIR AAI.\n\n\nSoftware and data availability\n\nNo data is needed to use the Authorization service.\n\nThe code is open source at: https://gitlab.com/uit-sfb/AuthService2.\n\nArchived code at time of publication is: https://doi.org/10.5281/zenodo.10589952\n\nThe software is licensed under The MIT License.\n\nA user guide is at: https://gitlab.com/uit-sfb/AuthService2.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFunding was provided from ELIXIR and ELIXIR-Norway. ELIXIR received funding from the European Union’s Horizon 2020 research and innovation program (ELIXIR- EXCELERATE, grant agreement 676559). ELIXIR-Norway is funded by the Research Council of Norway through the ELIXIR.NO project (grant number 208481).\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\nRobertsen EM, Kahlke T, Raknes IA, et al.: META-pipe - Pipeline Annotation, Analysis and Visualization of Marine Metagenomic Sequence Data. ArXiv160404103 Cs. 2016. Reference Source\n\nRaknes IA, Bongo LA: META-pipe authorization service (Version Tag: Zenodo-F1000). Zenodo. 2017. Data Source" }	[ { "id": "29637", "date": "22 Jan 2018", "name": "Mikael Linden", "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 paper is a useful description on how Meta-Pipe relies on OAuth2 framework for authorising access to the Meta-Pipe storage and job queue.\nWhat is the criteria the authorisation server uses to authorise the user to a certain resource? Based on what criteria is the authorisation granted to a user? Is it that users can only access their own files? Or are there also other criteria e.g. who in general can access the META-Pipe?\nELIXIR AAI provides an OpenID Connect provider that is an OAuth2 authorisation server. Could you use it instead of developing your own server?\nDoes the Meta-Pipe web application run in a user's browser or in a server?\nWhat is the relation of Feide and ELIXIR AAI in the context of Meta-Pipe? What is the relation of Meta-Pipe authorisation server and Feide? Does the Meta-Pipe authorisation server rely on them both for user authentication?\nIn the end of Standards section on page 4 you refer to RFC 7522 as a spec you used but on the previous page you say you couldn't use that spec.\nYou could improve figure 1 by clearly indicating where are the OAuth2 authorisation server, client and resource server. Perhaps you could also introduce another drawing where the OAuth2 messages are presented. That would make it easier for the reader to follow how you have mounted OAuth2 for your deployment.\nClients other than Meta-pipe Web app are missing in the Figure 1. What is the service behind the Meta-pipe REST API (from other sections I learn they are the job submission/management and storage services)?\nIn the future work section; instead of displaying the user a URL with an embedded access token, why can't you protect the URI with OAuth2, triggering the client to obtain the Access token from the Authorisation server directly and then presenting it to the storage server.\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? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3677", "date": "01 Jun 2018", "name": "Lars Ailo Bongo", "role": "Author Response", "response": "Thank you for your insightful remarks. We have made the following improvements to our paper to address the raised concerns:> What is the criteria the authorisation server uses to authorise the user to a certain resource?> Based on what criteria is the authorisation granted to a user?> Is it that users can only access their own files?> Or are there also other criteria e.g. who in general can access the META-Pipe?We added an explanation in the “Use cases” that all users with an ELIXIR AAI supported account have access to (only) their files and job information in META-pipe.> ELIXIR AAI provides an OpenID Connect provider that is an OAuth2 authorisation server. Could you use it instead of developing your own server?We added a discussion of the ELIXIR AAI Open ID Connect to the introduction. It was not an option when we started our implementation. We still believe the simplicity of our server has some advantages for development and testing purposes. For example as an introduction to OAuth2 and ELIXIR AAI.> Does the Meta-Pipe web application run in a user's browser or in a server?We now specify in Use Cases; User login via META-pipe web application that the web application is run only in the browser.> What is the relation of Feide and ELIXIR AAI in the context of Meta-Pipe?> What is the relation of Meta-Pipe authorisation server and Feide? Does the Meta-Pipe authorisation server rely on them both for user authentication?We added explanations in Methods; Integration with ELIXIR-Norway Galaxy/ Feide that we must use Feide for Galaxy. We also describe how the Galaxy tool has an account on the Authorization service, and acts on behalf of all Feide users logged in Galaxy. The Authorization service therefore do not directly rely on both for user authentication.> In the end of Standards section on page 4 you refer to RFC 7522 as a spec you used but on the previous page you say you couldn't use that spec.It is correct that we do not use RFC 7522, so we have removed it from the list.> You could improve figure 1 by clearly indicating where are the OAuth2 authorisation server, client and resource server. Perhaps you could also introduce another drawing where the OAuth2 messages are presented. That would make it easier for the reader to follow how you have mounted OAuth2 for your deployment.> Clients other than Meta-pipe Web app are missing in the Figure 1. What is the service behind the Meta-pipe REST API (from other sections I learn they are the job submission/management and storage services)?We have added an explanation to the figure caption that the web application is an OAuth2 Client, the META-pipe REST API is the OAuth2 resource server, and the Authorization Service is an OAuth2 authorization server. We chose not to make changes to the figure since we try to keep it simple for illustration purposes, and since more detailed figures for the OAuth2 messages can be found in the standard documentation.We also added a description to the figure caption that the META-pipe servers are represented by the REST API box.> In the future work section; instead of displaying the user a URL with an embedded access token, why can't you protect the URI with OAuth2, triggering the client to obtain the Access token from the Authorisation server directly and then presenting it to the storage server.Misunderstanding? Is in line with OAuth2 specification.We added an explanation in Future Work that the URI is built after the client obtains an access token from the Authorization service. We do not know of a better way to do this." } ] }, { "id": "31273", "date": "07 Mar 2018", "name": "Frank Oliver Glöckner", "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 agree with the authors that adding META-pipe to the ELIXIR AAI system is a useful invention. Since they were the first ones to add this feature to their pipeline I also appreciate the pioneering character of their work. To separate the META-pipe software from the authentication-server makes fully sense to me and supports the reusability of the authentication server for other projects.\n\nNevertheless, I have two main questions/concerns where I did not find the answer in the manuscript:\n\nSecurity: Although I am not an expert in this field I got the information that saving the Token in the local storage of the browser might be a risk. Since AAI is a security relevant component in every software system I would recommend consulting an expert in this field to check if the mode of implementation can be in general regarded as save. From scratch implementation: It is rather unclear for me why the authors have decided to go for a “from scratch” implementation of their authorisation server? On page two they briefly state that existing out-of-the box solutions did not fulfil all requirements or are too heavy weight for their application, but only OpenID Connect is given as an example. From our experiences several open source authentication-servers exist, that can do the job pretty well e.g. Keycloak, GLUU, hydra, Shibboleth Identity Provider, with the last one is already used in the ELIXIR environment. To make the advantages of a new implementation clear I would recommend adding a paragraph and a table where the different servers are compared and the reasons why going for a fresh implementation are explained in detail. Sticking with a well-established implementation might have also had advantages with respect to potential security issues. A rather comprehensive list of SSO implementations can be found here https://en.wikipedia.org/wiki/List_of_single_sign-on_implementations.\n\nIs the rationale for developing the new method (or application) clearly explained? No\n\nIs the description of the method technically sound? Partly\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? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3676", "date": "01 Jun 2018", "name": "Lars Ailo Bongo", "role": "Author Response", "response": "Thank you for your comments and remarks. We have improved the paper as follows:> I got the information that saving the Token in the local storage of the browser might be a risk. Since AAI is a security relevant component in every software system I would recommend consulting an expert in this field to check if the mode of implementation can be in general regarded as safe.We could not find clear guidelines for the where to store the access token and refresh. It does not seem unusual to store the token in the browser local storage. We agree that this may expose our Authorization server to some attacks (such as cross-site attacks). We have therefore added a comment to the Design section that this may be unsafe, and we are considering alternatives for our server.> It is rather unclear for me why the authors have decided to go for a “from scratch” implementation of their authorisation server? We have added a discussion of the ELIXIR AAI Open ID Connect, Shibboleth, and Keycloak in the introduction. It was not an option when we started our implementation, but is clearly an alternative today. We still believe the simplicity of our server has some advantages for development and testing purposes. In addition, we use many libraries for the implementation such as Spring security. We also believe the simplicity, combined with this paper, makes it a good introduction to OAuth2 and ELIXIR AAI." } ] } ]	1	https://f1000research.com/articles/7-32
https://f1000research.com/articles/7-687/v1	01 Jun 18	{ "type": "Research Article", "title": "Prevalence of intestinal parasites among food handlers attending public health laboratories in Khartoum State, Sudan", "authors": [ "Tarig A. Gamar", "Hassan H. Musa", "Hisham N. Altayb", "Mogeeb Kabbashi", "Yassen Alsayed", "Adam D. Abakar", "Hassan H. Musa", "Hisham N. Altayb", "Mogeeb Kabbashi", "Yassen Alsayed", "Adam D. Abakar" ], "abstract": "Background: Infections by intestinal pathogens especially protozoans and helminths are considered to pose a real health problem, particularly in the tropics. They cause considerable morbidity and mortality rates in developing countries. The high prevalence of these infections is closely correlated with poverty, poor environmental hygiene, and impoverished health services. This study aimed to detect prevalence and frequency of parasitic infections among food handlers in Khartoum Sudan.\n\nMethods: Three hundred and fifty Food-handlers, attending public health laboratories in Khartoum, Sudan, for an annual medical check-up, were screened for intestinal parasites by four laboratory techniques viz. direct faecal examination, formal-ether concentration, Baermann technique and agar culture method. Results: The infection rate was 23.7% by Formol-Ether Concentration technique, followed by direct saline stool preparation (7.1%). Out of 83 positive samples the infection rate among different nationalities was as follows: Sudanese 68 (81.9%), Ethiopians 13 (15.7%), Syrians 2 (2.4%) and Egyptians 0 (0%). Intestinal parasites were more prevalent among males (73; 25.1%) than female food handlers (10; 16.9%). Three protozoans, nematodes, two tap worms and one trematode worm were detected among infected population: their frequency were as follows: Entamoeba histolytica (7.4%), Entamoeba coli (6.86%), Giardia lamblia (6%), Schistosoma mansoni (1.40%), Necator americanus (1.43%), Hymenolepis nana (0.68%), Strongyloides stercoralis (0.68%), Taenia saginata (0.57%), Ascaris lumbricoides (0.57%) and Trichostrongylus species (0.29%). Conclusion: The overall prevalence of protozoan infections among food handler in Khartoum state, Sudan was 20.26% while the helminthic infections was 5.97%. Formol-ether concentration technique is better for detection of intestinal parasites than the direct faecal smear technique. Likewise, Barmann’s technique confirms detection of nematodes worms especially hookworms.", "keywords": [ "Food-handlers", "Baermann technique", "Formal Ether", "Agar Culture", "Sudan." ], "content": "Introduction\n\nIn developing countries, intestinal parasitic infections remain a significant cause of mortality and morbidity1,2. Parasitic infections can cause growth delay, iron deficiency anaemia, especially in children, and other psychological and physical health conditions3. The high prevalence of these infections is closely associated with poverty, penurious health services, and poor environmental and personal hygiene4.\n\nSoil-transmitted helminth (STH) and other helminth parasites like Taenia saginata and Hymenolepis nana, represent important causative agents of gastrointestinal infections. Intestinal protozoan dwellers, mainly Giardia lamblia and Entamoeba histolytica, also, contribute to intestinal disorders1.\n\nPolluted soil and water sources, and poor personal hygiene are the major factors in the transmission of parasitic infections to humans through the fecal-oral route5. Contaminated food caused by inadequate environmental sanitation and insufficient personal hygiene by food-handlers have been implicated in epidemics of protozoan infections in humans6. Approximately 500 million people worldwide are diagnosed with amoebiasis, with an annual mortality between 40,000 and 110,000 according to the World Health Organization (WHO)7. In 1978 in Geneva the WHO scientific group on the changing pattern of food hygiene problems underlined that many of the hazards related with microbial or parasitic contamination had reduced because of the intensive efforts of food hygiene services and producers8. Parasitic infections in food-handlers, which are often asymptomatic, can cause a real threat to immune-compromised patients9. According to the policy of Sudan ministry of health, food-handlers should be screened annually for parasitic infections. The objective of this study is to determine the infection rate and study distribution of intestinal parasite among Sudanese food-handlers in Khartoum, Sudan.\n\n\nMethods\n\nThis was a descriptive cross-sectional study conducted in Khartoum state in the central part of Sudan, during the period from October 2016 to April 2017. Khartoum state is located between longitudes 31.5–34°E and latitudes 15–16°N with an area of approximately 22.142 km2 (Figure 1) with a total population of about six million (see City Population site for Sudan). Stool samples were collected from public health laboratories (the public health lab in the Medical Commission) of Khartoum State; Omdurman locality, Khartoum North locality and Khartoum locality. The samples were analyzed at the Department of Parasitology, University of Science and Technology.\n\nA total of 350 stool samples were collected from food-handlers who attended for annual check-ups during the study period About 101 stool samples were collected from participants in the public health lab in Medical Commission in Khartoum North locality, 160 stool samples were collected from participants in the public health Lab in Medical Commission in Omdurman locality and 89 stool samples were collected from participants in the public health Lab in the Medical Commission in Khartoum locality. The sample size was calculated using this formula10.\n\nn= Z2PQ/d2 or N = Z2 P (p–1)/ d2\n\nWhere n = sample size\n\nP = prevalence rate\n\nZ = 1.96 at α = 0.05 (α = desired confidence level)\n\nd = desired width of confidence (precision)\n\nQ = 100-P\n\nThere for the sample size (n) was determined as:\n\n1.962×.05×.05102\n\nn = 96.04 ≈ 96\n\nN = (1.95) ² × (100 - 50) /5² = 380\n\nN= 380\n\nFour different methods were used for examination of stool samples: direct faecal examination, the formal–ether concentration technique, Barmenn’s apparatus technique and Agar culture method.\n\nDirect faecal examination. Direct microscopic (Olympus CX22 Microscope, Japan) examination of the sample was carried out in a systematic manner using a 4× objective lens to select the area to be screened, followed by a 10× objective lens to locate any parasitic objects. Suspicious objects were identified under a 40× objective lens. Then 2 drops of Lugol solution (Cat.No:09.0004.0500, dop®, Turkey) were added to facilitate identification of undifferentiated protozoan cysts and specimens were re-examined 5 minutes later1.\n\nFormal–ether concentration technique. The formal-ether concentration technique was performed by adding 1 g of faeces to 5 mL of formalin (10%), which was emulsified and strained, and the filtrate centrifuged for 2 min at 3000 rpm. Then 1 mL of sediment faeces and 9 mL of 10% formalin solution (cat no. F-04202, Oxford laboratory reagent, India,) were added to 3 mL of ethyl acetate and centrifuged further for 2 min at 2000 rpm. The upper 3 layers were decanted by inverting the tube and the last drop was allowed to fall back into the tube. Next, the filtrate was allowed to sediment by gravity for 15 min, prepared, examined and identified as in the direct smear technique1.\n\nBarmenn’s apparatus technique. Baermann’s technique was performed as described Garcia & Bruckner11 by adding 5 g of fresh faeces placed in the bottom of the strainer. The strainer was placed in the funnel. Warm water (40° C) was added to cover the faeces in the strainer. It was left undisturbed for 1–2 hours to give time for Strongyloides larvae to emerge from faeces into the water. The tip of the tube was opened and 7–10 ml of the fluid was collected into a centrifuge tube. A plastic bulb pipette was used to discard the suspension fluid into a container of disinfectant. The sediment was transferred to the slide and covered with a coverslip and was examined for motile larvae using 10× magnification11.\n\nAgar culture method. The procedure of agar plate culture was performed by adding 2g of fresh stool into the center of agar plate. Area approximately 1 in diameter was placed.\n\nThe lid was replaced and the plate was sealed with cellulose tape. The agar plate was maintained (right side up) at room temperature for 2 days. The sealed plates were examined after 2 days through the plastic lid under the microscope for microscopic colonies that develop as random tracks on the agar, indicating larvae at the ends of tracks away from the stool. A hole in the top of plastic petri dish was made with hot of forceps. 10 ml of 10% formalin was added gently through the hole onto the agar surface and swirled to cover the surface. The agar plate was then rinsed and allowed to stand for 30 min. The tape and lid of agar plate were removed. The 10% formalin was poured through a funnel into a centrifuge tube. The formalin rinse fluid was centrifuged for 5 min at 500xg. A wet smear preparation was prepared from the sediment and was examined at the 10× objective (low power) for the presence of larvae if larvae were found they were then identified with a 40× objective (high dry power)11.\n\nThe data were analyzed using SPSS (21.01) and Microsoft Excel 2010. Descriptive statistics for categorical data were formulated as frequency and percentage. Chi-Square test was used to compare of categorical data, while independent sample t-test was used to compare of numerical data. The significance level was considered at P-Value of < 0.05.\n\nSamples were collected from participants after explanation of the importance of the study and signing the consent form. The ethical approvals were obtained from the National Committee for research, at the ministry of health. Results of direct wet preparation of samples collected were donated for treatment of all participants included in the study and some results were dispatched to a physician for treatment prescription.\n\n\nResults\n\nThe total number of screened food-handlers included in the study was 350; the age of participant ranged from 16 to 68 years with an average age of 32 years, 46% of the participants were less than 29 years old compared with 54% of being 29 or above (Figure 2). The majority of participants were males (83.1.9%) and (16%) were female (Figure 3).\n\nDistribution of samples according to the residence data showed that 101 participants were from Khartoum north (28.9%), 160 participants were from Omdurman (45.7%) and 89 participants were from Khartoum (25.4%) (Table 1). The majority of the participants were Sudanese (83.1%), followed by the Ethiopian (13.4%), Syrian (3.1%) and the Egyptian (0.03%) (Figure 4).\n\nDirect microscopy detected intestinal parasitic infection in 7.1% of the food-handlers, by using the Formal-Ether Concentration technique (the most sensitive) the study found intestinal parasite's infection 23.7% of the food-handlers. The result of Baermann’s technique was used to detect larva of Strongyloides stercoralis and Hookworm (Figure 5). The Agar culture method show in (Table 2). Parasitic infection in stool samples were found by the Formal-ether and direct saline stool preparation, the most prevalent protozoan parasite was Entamoeba coli and for helminthic parasites was Ascaris lumbricoides in Khartoum north locality (Table 3). The most prevalence protozoan parasite in Omdurman locality was Entamoeba coli (9.4%) and for helminth parasites Schistosoma mansoni (1.87%) (Table 4). The most prevalence protozoan parasite in Khartoum locality was Giardia lamblia (3.3%) and the most prevalent helminth infection was Necator americanus (3.3%) (Table 5). The overall prevalence of parasitic infections among food handler in Khartoum state are demonstrated in Figure 6.\n\nResidence and occupation were found to have a significant association with the result of the direct wet examination. The OR indicated that those respondents who working in restaurants were 2.25 times more likely to have positive test compared with others (Table 6). Occupation was found to have a significant association with the result of formal ether and the OR indicated that those respondents who working in restaurants were 4.23 times more likely to have positive test compared with others (Table 7). The infection rate among different nationalities was as follows: Sudanese 68 (81.9%), Ethiopians 13 (15.7%), Syrians 2 (2.4%) and Egyptians 0 (0%) (Figure 7). Intestinal parasites were more prevalent among males 73 (25.1%) over female food handlers 10 (16.9%) (Figure 8).\n\n\nDiscussion\n\nFor this study we select a descriptive cross-sectional and analytical facility bases study to be conducted in public health lab(s) of Khartoum State. The study was conducted between the years 2015 and 2018. This study is subject to several limitations, gastrointestinal parasitic infections are frequently reported among different sectors in Khartoum state especially in the territories of the town where hygienic conditions are poor. Most of the targeted participants do not seek any medical or health advice unless they have been to enforce to do so during routine annual medical chick up. This study found that formal-ether concentration technique is the most sensitive method for diagnosing the intestinal parasites. The level of infection with intestinal helminthes in food-handlers in Khartoum state, Sudan (5.97%) was much more than that reported by Babiker et al. who found that 2.7% of Sudanese food-handlers were infected with intestinal helminths9. It also higher than that reported by Tomaso et al. (0.2%) among food-handlers in Austria12. This may be the result of the migration of foreign nationals to\n\nthe capital, migration of individuals from the rural areas to urban areas, lack of personal hygiene, poor management of waste and poor sanitization. In food-handlers of Khartoum Sudan, the infection of intestinal helminths was much lower than non-Jordanian food-handlers working in Jordan as described by Al Lahham et al. who found that 13.5% of them were infected with intestinal helminthes13. It is also much lower than the infection rate for intestinal helminthes (18.3%) that was described by Costa-Cruz et al. in Brazil14.\n\nThe intestinal protozoal infection ratio of our study was low (20.26%) among food handlers in Khartoum State, Sudan. This finding was strongly supported by similar study conducted In Northwest Ethiopia, 29.1%5.\n\nOur study has a low infection rate when comparing with the study conducted in 2009 by Babiker et al. (29.4%)9. This could be attributed to difference of subjects participated in the two studies. For this study, examining Ethiopians, Egyptian and Syrians gave a very low infection rate that affects the overall prevalence. Similar variation was also reported when comparing this study with other research done else were, for example, in Jordan, the frequency of infection by intestinal protozoal was 30.2%13.\n\nThe current study revealed that Infection with G. lamblia was lower than that reported in Somalia (77%)15.\n\nFurthermore, a similar study in Jordan presented by Al-Lahham et al.,13 showed that the high rate of infection by intestinal protozoal (30.2%) which is higher than our conducted study. It has been previously reported that about 15% of food-borne disease epidemics are the consequence of infection by food-handlers16. 6.5% of food handlers in Bahir Dar town in Ethiopia had protozoal and helminthic infection17. Infected food-handlers have been involved in parasitic transmission, resulting in an epidemic in food eateries with poor hygiene posing hazards to travelers18,19. Not all of the participants had access to proper health education on food safety, although education levels could have predisposed the arrangement of infection20. Numerous food-handlers did not give the impression of being aware of simple safety and health necessities to work with food and food products. The economic situation may have impact in food production and it can be affected by the foodborne disease, hence large amounts of emolument may be lost by individuals due to reduced efficiency and costs on medical care21. Multi-sectoral attitude to food safety at all levels cannot be overstated22.\n\nIn our study, a relationship was found in parasitic infection with occupation and residence, but no association was found between the frequency of parasite infection and age, gender, and nationality. We find that the annual monitoring of food-handlers in Khartoum is insufficient to screen parasitic infections and that more frequent screening, for example, monthly screening, should be supported. Our recommendations are that health education and personal hygiene should be included in the annual check of food handlers. The use of the most sensitive technique for diagnosing intestinal parasitic infections should be established.\n\n\nData availability\n\nDataset 1: Demographic and parasitic infection data for participants http://doi.org/10.5256/f1000research.14681.d20480023", "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 extend their heartfelt thanks to the participants and staff in the public health Lab for their support.\n\n\nReferences\n\nCheesbrough M: Medical laboratory manual for tropical countries vol11-microbiology. 1984. Publisher Full Text\n\nWorld Health Organization: Prevention and control of intestinal parasitic infections: report of a WHO Expert Committee. [meeting held in Geneva from 3 to 7 March 1986]. 1987. Reference Source\n\nGordon N: Iron deficiency and the intellect. Brain Dev. 2003; 25(1): 3–8. PubMed Abstract \| Publisher Full Text\n\nAl-Braiken FA: Is intestinal parasitic infection still a public health concern among Saudi children? Saudi Med J. 2008; 29(11): 1630–1635. PubMed Abstract\n\nAndargie G, Kassu A, Moges F, et al.: Prevalence of bacteria and intestinal parasites among food-handlers in Gondar town, northwest Ethiopia. J Health Popul Nutr. 2008; 26(4): 451–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChute CG, Smith RP, Baron JA: Risk factors for endemic giardiasis. Am J Public Health. 1987; 77(5): 585–587. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuaces AL, Osorio LM, Barrett AJ: A new test for infection by Entamoeba histolytica. Parasitol Today. 1993; 9(2): 69–71. PubMed Abstract \| Publisher Full Text\n\nMatyáš Z: Role of veterinarians in modern food hygiene. Bull World Health Organ. 1978; 56(5): 699–711. PubMed Abstract \| Free Full Text\n\nBabiker MA, Ali MS, Ahmed ES: Frequency of intestinal parasites among food-handlers in Khartoum, Sudan. East Mediterr Health J. 2009; 15(5): 1098–104. PubMed Abstract\n\nSathian B, Sreedharan J, Baboo SN, et al.: Relevance of sample size determination in medical research. Nepal J Epidemiol. 2010; 1(1): 4–10. Publisher Full Text\n\nGarcia LS, Bruckner DA: Diagnostic medical parasitology. American Society for Microbiology (ASM). 1997. Reference Source\n\nTomaso H, Dierich MP, Allerberger F: Helminthic infestations in the Tyrol, Austria. Clin Microbiol Infect. 2001; 7(11): 639–641. PubMed Abstract \| Publisher Full Text\n\nAl-Lahham AB, Abu-Saud M, Shehabi AA: Prevalence of Salmonella, Shigella and intestinal parasites in food handlers in Irbid, Jordan. J Diarrhoeal Dis Res. 1990; 8(4): 160–162. PubMed Abstract\n\nCosta-Cruz JM, Cardoso ML, Marques DE: Intestinal parasites in school food handlers in the city of Uberlândia, Minas Gerais, Brazil. Rev Inst Med Trop Sao Paulo. 1995; 37(3): 191–196. PubMed Abstract \| Publisher Full Text\n\nIlardi I, Sebastiani A, Leone F, et al.: Epidemiological study of parasitic infections in Somali nomads. Trans R Soc Trop Med Hyg. 1987; 81(5): 771–772. PubMed Abstract \| Publisher Full Text\n\nRosenfield PL, Golladay F, Davidson RK: The economics of parasitic diseases: Research priorities. Soc Sci Med. 1984; 19(10): 1117–1126. PubMed Abstract \| Publisher Full Text\n\nAbera B, Biadegelgen F, Bezabih B: Prevalence of Salmonella typhi and intestinal parasites among food handlers in Bahir Dar Town, Northwest Ethiopia. Ethiop J Health Dev. 2010; 24(1). Publisher Full Text\n\nMintz ED, Hudson-Wragg M, Mshar P, et al.: Foodborne giardiasis in a corporate office setting. J Infect Dis. 1993; 167(1): 250–253. PubMed Abstract \| Publisher Full Text\n\nWolfe MS: Giardiasis. Clin Microbiol Rev. 1992; 5(1): 93–100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAngelillo IF, Viggiani NM, Rizzo L, et al.: Food handlers and foodborne diseases: knowledge, attitudes, and reported behavior in Italy. J Food Prot. 2000; 63(3): 381–5. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: World Health Day 2012: ageing and health: toolkit for event organizers. 2012. Reference Source\n\nLikar K, Jevšnik M: Pogoji za vzpostavitev učinkovitega notranjega nadzora. 2004.\n\nGamar TA, Musa HH, Altayb HN, et al.: Dataset 1 in: Prevalence of intestinal parasites among food handlers attending public health laboratories in Khartoum State, Sudan. F1000Research. 2018. Data Source" }	[ { "id": "35563", "date": "23 Jul 2018", "name": "Mubarak M. Abdlrahman", "expertise": [ "Reviewer Expertise Parasitology", "Molecular biology", "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\nGood simple study highly needed for the country like Sudan. The work is original and reflect the health situation of the foreign labor that working in the restaurants and food industry in Sudan. I have some comments on the paper:\nIn paragraph one it is Khartoum State, not just Khartoum. In the third paragraph of the Introduction if the Author change the word polluted with the word contaminated is better. In Table 3 if the Author use the word sentence percentage in Khartoum North in the 3ed upper cell of the table is better. And the sentences percentage in Khartoum State in the 4th upper cell is also better. In Table 4 if he delete the word (the) from the upper cells is better. Figure 6 should come before Table 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? 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": "36648", "date": "20 Aug 2018", "name": "Guéladio Cissé", "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 paper addresses the very interesting topic of food borne diseases risks in developing countries. It presents an estimation of the prevalence of intestinal parasites among a specific exposed group in the food chain (the handlers).\nThe literature is not enough recent. An effort could be made by the authors to search for some more recent studies on the same topic.\nThe study needs further clarifications at the level of Methods(page 3):\nUsing the term \"a cross-sectional study\" and indicating a period between October 2016 and April 2017 (6 months) is questionable. Can a background on the season(s)over the period be provided?\nSampling and sample size (page 3)\n\nIf it is the team itself that did all the examinations with the same 4 standardized laboratory methods applied to samples from the 3 indicated laboratories, then the calculated (expected) sample size should be presented first and then the strategy to reach the number from the 3 centers be presented. How to reach the sample size in a rigorous and balanced way between 3 targeted laboratories?; Are there any exclusion criteria? Was there a limit of days or weeks during which the number of handlers to enroll should be reached? How is the team alerted about a food handler coming to the screening of a laboratory?...If possible, more explanation on the selection process could be interesting. It is better to not present in the first sentence the effective sample size reached, but rather provide the number under Results\n\nMethods of samples examination\nAfter being presented all the 4 methods, the reader may expect a clear indication of what method has been found more appropriate for which result;\n\nResults\nEffectively more appropriate to indicate here the final number of handlers who successfully provided samples. A table could be helpful to present the major results from each method Please complete all the titles of figures and tables, to make sure that each title is self standing. Example: Figure 2 should better read like this: \"Samples of food handlers distribution according to age in Khartoum, (October 2016 - April 2017)\". Please do this for all titles More specifically for Figure 6 (page 7), maybe helpful to recall in the title the methods used for detecting these parasites. Figure 7: Not very much sure that this is reflecting the real percentage of infection among the different nationalities. If the figure is about the distribution of the total number of cases between the different nationalities, it does not make much sense as the Sudanese group is far much larger; it would be much more interesting to present the % among the same group (number of cases of the group/total number of the group); with this the figure could be completely something else; please check this; maybe a statistician can assist also on this\n\nDiscussion\nThe first sentence is a little bit confusing. Maybe \"We presented a descriptive cross sectional study ...\" And therefore you should avoid a repetition of \"study\" in the sentence. Please rework this sentence. The second sentence is also with an error about dates; under Methods it was stated between October 2016 and April 2017,here it is now stated \"... between years 2015 and 2018\". Please correct or clarify. \"This study is subject to several limitations\". Where are they presented? Please state them clearly. \"gastrointestinal parasitic infections ...\" this sentence is starting without a capital letter and it is seems completely disconnected from the sentences above. please check this A clear limitations paragraph is needed\n\nConclusion\nA clear conclusion paragraph is needed.\n\nEnglish\nThere are a few grammatical corrections needed, and an additional careful reading may be helpful\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?\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-687
https://f1000research.com/articles/7-139/v1	01 Feb 18	{ "type": "Research Article", "title": "Alternative splice variants of rhomboid proteins: In silico analysis of database entries for select model organisms and validation of functional potential", "authors": [ "Joshua Powles", "Kenton Ko", "Joshua Powles" ], "abstract": "Background: Rhomboid serine proteases are present in many species with sequenced genomes, and are often encoded in each species by more than one predicted gene. Based on protein sequence comparisons, rhomboids can be differentiated into groups - secretases, presenilin-like associated rhomboid-like (PARL) proteases, iRhoms, and “inactive” rhomboid proteins. Although these rhomboid groups are distinct, the different types can operate simultaneously. Studies in Arabidopsis showed that the number of rhomboid proteins working simultaneously can be further diversified by alternative splicing. This phenomenon was confirmed for the Arabidopsis plastid rhomboid proteins At1g25290 and At1g74130. Although alternative splicing was determined to be a significant mechanism for diversifying these two Arabidopsis plastid rhomboids, there has yet to be an assessment as to whether this mechanism extends to other rhomboids and to other species. Methods: We thus conducted a multi-year analysis of databases to determine if the alternative splicing mechanism observed for the two Arabidopsis plastid rhomboids was utilized in other species to expand the repertoire of rhomboid proteins. To help verify the in silico findings, select splice variants from different groups were tested for activity using transgenic- and additive-based assays. These assays aimed to uncover evidence that the selected splice variants display capacities to influence processes like antimicrobial sensitivity. Results: The multi-year in silico assessment for six model experimental species (human, mouse, Arabidopsis, Drosophila, nematode, and yeast) revealed robust usage of alternative splicing to diversify rhomboid protein structure across the various motifs or regions, especially in human, mouse and Arabidopsis. Subsequent validation studies uncover evidence that the splice variants selected for testing displayed functionality in the different activity assays. Conclusions: The combined results support the hypothesis that alternative splicing is likely used to diversify and expand rhomboid protein functionality, and this potentially occurred across the various motifs or regions of the protein.", "keywords": [ "Rhomboid proteins", "alternative splicing", "activity", "transgenic expression", "recombinant rhomboid proteins", "protein structure", "model organisms", "databases" ], "content": "Introduction\n\nRhomboid proteins are found widely in all types of organisms. In higher-order organisms, rhomboid proteins are often encoded by a large group of genes1, for example, upwards of twenty-two database entries for Arabidopsis and thirteen for humans (assessed as of May 2017). Rhomboids are also part of an even larger group of proteins involved in regulated intramembrane proteolysis2–31. So far, rhomboid genes can be divided into two subgroups encoding proteolytically active secretases- and Presenilin Associated Rhomboid Like (PARL)-type rhomboid forms, and two subgroups of catalytically inactive forms (“non-proteases”) such as iRhoms, Derlins, and other distantly related forms1, 26–29.\n\nThe ‘active’ category of rhomboid proteases are often found to occupy regulatory roles of different cellular activities, typically by interacting with and cleaving specific membrane-residing substrates. Examples being the epidermal growth factor signaling pathway in fruit flies32, the quorum-sensing mechanism of the bacterium Providencia stuartii33–34, yeast mitochondrial membrane remodelling35, and the health status of mitochondria in human cell lines36.\n\nThe ‘inactive’ subcategories of rhomboid proteins are believed to lack the needed catalytic residues used by the active rhomboid proteases1. Despite the absence of the catalytic residues, some of the inactive rhomboid proteins are found to be functionally significant without being active proteases32–35,37. In some of the reported cases, interaction alone with an inactive rhomboid, without proteolysis, is sufficient to cause effects, such as growth signaling in cancer cells (human iRhom1), dislocation of proteins in the endoplasmic reticulum protein (ER) (mammalian Derlins), ER protein quality control (Drosophila iRhom Rhomboid-5), development, and organelle biogenesis (Arabidopsis At1g74130)26–29,37–43. Even the active human rhomboid protease, RHBDL4, a promoter of ER-associated degradation of membrane proteins, physically interacts with ubiquitin in order to proceed with its protease activities44.\n\nIn addition to the subcategories of active and inactive rhomboid proteases, alternative splicing appears to generate even more variants with modified functionalities1. For example, one case was confirmed for the human RHBDD2 gene45. Levels of the two alternatively spliced RHBDD2 mRNAs were elevated in breast cancer cell lines41, suggesting a link between cancer cell activity, and the presence of splice variants. In Arabidopsis, the active plastid rhomboid At1g25290 was confirmed to exist as two functionally significant splice variants that differ by the presence of a potential cyclin-binding motif, a motif known to be involved in cell cycling46. One of the inactive plastid rhomboids predicted for Arabidopsis, At1g741301, also exists as three splice variants, with distinct functionalities and different levels of interactions with the Tic40 substrate47. Functionality of the three At1g74130 splice variant proteins was apparent upon testing at the whole cell level in bacteria and yeast. Despite being plant-derived, the At1g74130 splice variants exhibited physiological interactions with the mitochondrial rhomboid protease Rbd1 in yeast cells, and modulated the cleavage ratio of the resident mitochondrial protein Mgm1, a ratio that governs mitochondria remodeling and respiratory status47.\n\nTo date, the phenomenon of diversifying rhomboid protein functionality through alternative splicing has been documented for two Arabidopsis plastid rhomboids. It has not yet been assessed as to whether this phenomenon is limited, or represents a mechanism for expanding the number of functional rhomboid forms in a wide range of rhomboid systems and organisms. Therefore, using the findings reported for At1g25290 and At1g74130 as guidance, we carried out a periodic analysis of genetic databases of model experimental species to determine the possible extent of alternative splicing in rhomboid genes and, based on coding RNA sequence entries, how splice variants may be reflective of a way to diversify functionality. A limited selection of alternatively spliced variants were then analyzed to document evidence of potential activity using different types of assays.\n\n\nMethods\n\nThe sequence entries used were assembled from current versions of the publicly-accessible databases (last sampled as recent as May 31, 2017). The databases contained both validated and currently unverified entries. We further analysed all available ESTs as verification of the different sequences. In the end, both types of entries were analyzed. We used the NCBI database (RefSeq, RRID:SCR_003496) as our primary source for the selected model organisms (Homo sapiens (human), Mus musculus (mouse), Arabidopsis thaliana (Arabidopsis), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (nematode) and Saccharomyces cerevisiae (Baker’s yeast). We also cross-checked with other databases such as the Mouse Genome Informatics resource (Mouse Genome Informatics, RRID:SCR_006460), TAIR for Arabidopsis (TAIR, RRID:SCR_004618), FlyBase (FlyBase, RRID:SCR_006549), WormBase (WormBase, RRID:SCR_003098), and the Saccharomyces Genome Database (SGD, RRID:SCR_004694). All entries used are compiled in Table S1 along with their relevant details. Table S2 provides a listing of formal and alternate names used for each rhomboid, along with Gene ID numbers and related references. For this study’s objective of periodically analyzing alternatively spliced products and their potential functionality, any more recent database updates are not expected to impact the conceptual findings.\n\nDatabase entries were compiled and assessed for alternative splicing before use. This was necessary to acquire sequences that resulted from alternative splicing only, as opposed to derivations from other routes. These assessments utilized all sequences deemed relevant, independent of the validation status indicated. This analysis required multiple comparisons between entries and genomic sequences, then converted into protein sequences, compared, and aligned with current models of rhomboid proteins. All variant RNA sequences were assumed to result in the generation of functional proteins, including extensively truncated products. The resulting predictions did not indicate that this assumption would not hold true. Any changes in the entries encountered in the periodic rounds of searching were assessed before updating our analyses. A prime example of such a situation occurred with the human database entries, where the newest version contained more verifications than the previous versions.\n\nThe categorization of alternative splicing events/splice variants was based on the potential impact of such changes on motifs along the protein, from amino to carboxyl terminus. The protein models and motifs used for categorization were adapted from the models reported by Lemberg and Freeman1. In many cases, alternative splicing events tend to influence one motif, but there were a number of cases where the impact may affect one or more motifs, depending on the type and location of the splicing event. For this study, sequences and splicing events are categorized at the motif-specific or region-specific level, and may thus appear in more than one category. For instance, a frequent occurrence is an alternative splicing event which occurs in a particular motif that would likely impact the adjacent linker region. The various splice variants are listed in Tables S3 to S10 by category. Information concerning the impact of the splicing events is also included in these Supplementary Tables. Overviews are provided in the Results section. Specific examples are highlighted when applicable.\n\nStructural predictions were facilitated by comparing alignments with predicted structures constructed and reported by Lemberg and Freeman1. Three-dimensional predictions of splice variant protein structures were created and compared by utilizing Phyre Version 2 services (RRID:SCR 010270)48 and visualized using PyMol 1.1 (RRID:SCR 000305). These 3D predictions were carried out solely for the purpose of investigating and speculating the capacity for structural impact of the different splicing events. All of these 3D predictions are provided and reported in the Supplementary Material for consideration only and do not represent established, solved structure data.\n\nSelect proteins were synthesized in Escherichia coli JM109 (DE3) and facilitated by the T7 promoter of pET20b (EMD4 Biosciences, Rockland, MA, USA). Histidine tags were joined in-frame to the carboxyl-terminus. Cells were grown at 16°C in ampicillin-containing Terrific Broth (Bioshops Inc., Burlington, Canada). Recombinant histidine-tagged proteins were purified using nickel – nitriloacetic acid affinity chromatography (Qiagen, Toronto, Canada). The protein expression and chromatography procedures followed were as reported and cited previously47. Proteins were quantified using the Bradford assay system (Biorad, Hercules, CA, USA) and normalized before use.\n\nProtein samples, typically 0.5 μg per lane, were analyzed when needed using standard one-dimensional (1D) 12% (m/v) sodium dodecyl sulfate (SDS) – polyacrylamide gels. Electrophoretic and immunoblotting protocols were performed according to Laemmli49 and Towbin et al.50. Immunoreactive bands were analyzed using scans, quantitated by densitometry (Scion Image 4.0.3.2, Scion Corporation, USA), normalized, and compared relative to internal references when applicable. All immunoblots were repeated at least three times within each experiment and with biological replicates. Representative results are then shown in the figures. When needed, quantitations were conducted using nonsaturated scans of the images presented in the figures. If used, the rabbit polyclonal anti-rhomboid protein (At1g74130) antibodies were established by our lab and validated as reported in Powles et al.51. For samples derived from transgenic yeast cell assays, rabbit polyclonal anti-yeast mtHsp70 antibodies (Antibodies-Online Cat# ABIN488515, RRID:AB_11209968) were used for normalization as reported previously in Powles et al.51. The normalized ratio strategy was designed to allow semi-quantitative assessment of profile changes independent of experiment, gel origin, and exposures. Statistical analyses or details are noted where applicable.\n\nActivity assays using transgenic yeast – Protein expression in S. cerevisiae (Baker’s yeast) was facilitated by the yeast – E. coli shuttle vector pACT2 (BD Biosciences-Clontech, San Jose, CA, USA). In all cases, expression of the inserted cDNAs was driven by the cloned yeast Rbd1 promoter. The introduction of plasmids was carried out using standard yeast transformation techniques. The host yeast strain C6000 used was acquired from EUROSCARF (EUROpean Saccharomyces Cerevisiae ARchive for Functional Analysis, RRID:SCR_003093) (Frankfurt, Germany). Cells were grown in glucose-supplemented media (at 30°C in glucose-supplemented yeast complete medium without leucine where applicable). All strains were prepared as populations (as opposed to from single colonies) and stored at -80°C as glycerol stocks.\n\nA disk diffusion method was employed to test different yeast strains for changes to nystatin sensitivity as a result of the indicated splice variant proteins being expressed. Yeast cells were suspended in top agar (1% w/v) media at 5 × 107 cells/mL and poured onto agar plates (2% w/v). Sterilized filter paper disks (38.5 mm2) infused with nystatin, were evenly positioned on the top agar, at a typical concentration of 10 disks per 90 mm plate. Each disk was infused with 10 µL of a 2.4% (v/v) nystatin solution, diluted with the appropriate yeast culturing medium. Plates were incubated overnight at 30°C. The zone of growth inhibition around each disk was then measured, and the corresponding area calculated. Multiple independent experiments were conducted to confirm the functional potential of the various splice variants.\n\nActivity assays using yeast and externally-added proteins - Non-transformed cells (without plasmids) were also assessed with exogenously added splice variant proteins. Since amphotericin B (AmB) has the ability to introduce transmembrane channels for protein delivery, cells were incubated for one hour with 10 µg (at 1 µg/mL) of the indicated recombinant variant protein along with a 1% (v/v) AmB solution (this level has no detrimental effect on yeast cell survival). Minimal volumes of cells, typically at 5,000 cells/mL, were used. Various levels of nystatin were then added and incubated for the last 15 minutes of the 1 hour treatment period. After the 1 hour incubation-treatment, 500 cells were plated (per 90 mm petri plate) and grown for 48 hours at 30°C to assess sensitivity to nystatin as a test for biological activity and for any differences between splice variants. Results from independent replicates were analyzed statistically (t-test) and noted with details where applicable.\n\nActivity assays using transgenic bacteria – E. coli JM109 (DE3) cells harboring various pET20b-based splice variant constructs were plated on LB agar containing varying concentrations of ampicillin. The assessment of ampicillin sensitivity was not dependent on induction of expression, but instead relied upon the inherent “leaky” expression. Plating was normalized with equal colony numbers. Results from independent experiments were analyzed statistically (t-test), and noted with details where applicable. Changes in ampicillin sensitivity were further examined using whole cell extracts and immunoblotting to assess β-lactamase expression, secretion, and processing of the precursor β-lactamase form.\n\nActivity assays using bacteria and externally added proteins - Bacteria (HB101) were assessed in some cases with exogenously added splice variant proteins. Cells harboring pET20b were used as the ampicillin resistance model for testing biological activity and differences between splice variant proteins. Dimethyl sulfoxide (DMSO) was utilized to permit the delivery of proteins into cells52 by an initial incubation of 30 minutes with 5% (v/v) DMSO, 10 μg of variant protein (normalized with elution buffer), and LB broth. After the 30 minute protein delivery period, each treatment was incubated with 1.25 or 1.5 mg/mL of ampicillin (and adjusted if needed) for an additional 45 minutes. Treatments were carried out at 37°C with shaking (100 rpm). Bacteria were normalized to 800 cells per treatment in a total volume of 200 μL, and plated in its entirety on LB plates and grown overnight at 37°C. Surviving colonies were counted and compared to mock treatments, which consisted of all components without proteins. Results from independent experiments were analyzed statistically (t-test) and noted with details where applicable.\n\n\nResults\n\nWe previously verified in separate studies a mechanism for diversifying rhomboid proteins and their functionality. This alternative splicing mechanism played diversifying roles for two different Arabidopsis plastid rhomboid genes - the active secretase type At1g25290 and the inactive PARL type At1g74130. Alternative splicing impacted different parts of the proteins with no apparent functional similarities to each other. The At1g25290 splice variants were focused on controlling the appearance of the cyclin-binding RVL motif in the protein’s middle segment, right after the third predicted transmembrane region46. The splice variants created for At1g74130 resulted in different shortened proteins, each missing a key glutamine residue in the last carboxyl transmembrane region47. Both studies provided evidence that the resulting variant proteins display altered functionality46,47. These two sets of findings alone bring the total number of plastid rhomboid forms in Arabidopsis to at least seven, two for At1g25290, three for At1g74130 and at least one each for At1g74140 and At5g25752.\n\nThe outcomes discussed above prompted us to look at rhomboid genes of other model species for evidence of similar diversification mechanisms. We thus periodically analyzed the RNA sequence databases of model organisms with complete genome sequences, human, mouse, Arabidopsis, Drosophila, C. elegans, and S. cerevisiae. The entries were compiled for use only after extensive EST analyses (the in silico verification process). Even though the databases continue to evolve, the observations disclosed here continue to be applicable. This notion remained intact even after repetitive assessments of the databases every six months since 2012.\n\nThe first aspect to establish was the presence of alternative splicing and its extent within a model species, and across the different selected model species. Using the current versions of the RNA sequence databases, we compiled all possible RNA sequence entries that were derived from alternative splicing, as opposed to other possibilities (see Methods for the sorting and verification process). The human, mouse, and Arabidopsis assessments revealed evidence of alternative splicing in different rhomboid genes of these species. There were 95 entries in human, 53 in mouse and 40 in Arabidopsis (Figure 1). In contrast, similar analyses of Drosophila, C. elegans and S. cerevisiae, revealed minimal levels to no evidence of alternative splicing. We found one possibility each for Drosophila and C. elegans and none so far for S. cerevisiae. It is, however, possible that the outcomes observed for the latter three model species were due to the limited number of reported alternate RNA sequences. The possibility of unreported alternative RNA sequences, even with limited variations, may help determine more definitively the presence and extent of alternative splicing in these species. This was the case in Arabidopsis where alternative splice variants for At1g25290 and At1g74130 were discovered and verified upon further analysis of transcript populations46,47.\n\nEach model organism is indicated along with the total number of rhomboid and rhomboid-like genes found in the databases and the number of accompanying variant sequences obtained and studied in this report. Entry details are summarized in Table S1. Both validated and unvalidated entry types were analyzed.\n\nOf the sequenced model species analyzed, the human rhomboid system appears to exhibit the most alternative splice variants. All 13 of the rhomboid or like genes display multiple entries reflective of alternative splicing (see Figure 2 and Table S1). For instance, human PARL contains verified splice variants and additional predicted mRNA sequences or proteins. Similar situations were observed for human RHBDF2 (iRhom2), RHBDL1, RHBDD1, and RHBDD2.\n\nEach region of the generalized rhomboid protein is indicated along with the total number of rhomboid and rhomboid-like genes that were accompanied by splice variants found in the databases. The genes considered belong only to the model organisms selected for study here and are tallied together independent of species. The total number of accompanying alternative splice variants determined for each region is also provided. These variants are again tallied independent of species. Entry details are summarized in Table S1. Both validated and unvalidated entry types were analyzed.\n\nThe use of alternative splicing is also evident in the mouse rhomboid genes. The assessment revealed evidence of multiple alternative splice variants for mouse rhomboids (Figure 1 and Table S1).\n\nCurrently, of the sequenced model genomes available, Arabidopsis appears to possess the highest number of rhomboid and like genes. There are 22 entries and 10 are accompanied by 1 or 2 additional splice variant sequences (Figure 1 and Table S1). The splice variants arising from At1g25290 and At1g74130 were discovered and verified in two other studies46,47. Based on the trends observed for human and mouse, it is likely that there are other splice variants in Arabidopsis awaiting discovery, especially for the other 12 gene entries currently without accompanying sequence variants in the database.\n\nFurther analyses of the Figure 1 entries indicate that many of the occurrences are likely reflective of mechanisms for diversifying functionality (Figure 2 and Table S3–Table S10). Structural changes were observed for both active rhomboid proteases (secretases and PARLs) and rhomboid-like proteins (inactive rhomboids and iRhoms) (Figure 3–Figure 6, Table S4–Table S9). Potentially impactful changes were located in domains found across the entire protein structure (Figure 2–Figure 6 and Table S3–Table S9). Changes were also observed within the 5’ UTRs that may affect translation and 3’ UTRs that may affect transcript properties (Tables S3 and 10). The changes impacting the protein can be subtle, affecting a few amino acid residues, to entire sections of the protein. In some instances, there were extensive deletions, insertions, truncations, or shortenings of the protein.\n\nAlternative splicing and resulting changes to the secretase-type rhomboid proteases (6+1 model). Amino acid sequences of the splice variant proteins were aligned and compared. The outcomes of these comparisons are compiled in the Supplementary Tables. The results are summarized in this figure. The regions of the protein potentially influenced by the changes are annotated. The protein structure is constructed using information from Lemberg and Freeman1. Information concerning the names used in the figure is provided in Table S2.\n\nAlternative splicing and resulting changes to the PARL-type rhomboid proteases. Amino acid sequences of the splice variant proteins were aligned and compared. The outcomes of these comparisons are compiled in the Supplementary Tables. The results are summarized in this figure. The regions of the protein potentially influenced by the changes are annotated. The protein structure is constructed using information from Lemberg and Freeman1. Information concerning the names used in the figure is provided in Table S2.\n\nAlternative splicing and resulting changes to iRhom proteins. Amino acid sequences of the splice variant proteins were aligned and compared. The outcomes of these comparisons are compiled in the Supplementary Tables. The results are in this figure. The regions of the protein potentially influenced by the changes are annotated. The protein structure is constructed using information from Lemberg and Freeman1. Information concerning the names used in the figure is provided in Table S2.\n\nAlternative splicing and resulting changes to “inactive” rhomboid proteins (using the 1+6 model). Amino acid sequences of the splice variant proteins were aligned and compared. The outcomes of these comparisons are compiled in the Supplementary Tables. The results are in this figure. The regions of the protein potentially influenced by the changes are annotated. The protein structure is adapted from Lemberg and Freeman1. Information concerning the names used in the figure is provided in Table S2.\n\nChanges to the 5’ untranslated region of the transcript - One of the most widely reported alternative splicing mechanisms is designed to control entry into translation or protein translation itself53. This continues to be the case for the rhomboid genes examined here (Table S3). Alternative splice variants with potential effects on translation were found in human, mouse and Arabidopsis. Twelve of the 13 human, and nine of the 13 mouse rhomboid genes were accompanied by entries with changes to the 5’ UTRs. Interestingly, despite the higher number of genes documented for Arabidopsis, 5’ UTR splice variants were found only for 7 of the 22 rhomboid genes. However, the absence of evidence for the other 15 Arabidopsis rhomboid genes may be due to unreported sequences or awaiting discovery.\n\nChanges to the amino terminal region - Alternative splicing events affecting the amino termini appear to be common as well in our set of RNA sequences (Figure 2–Figure 6 and Table S4). Changes affecting the region between the start methionine to the first predicted TMD are placed into this category which includes frame-shifts, alternate starting methionines, insertions and deletions. Changes in this region could affect functional aspects like protein targeting and transport, membrane insertion, assembly and topology, and assembly with complexes. With the exception of human RHBDD1, all of the other 12 human rhomboid genes are accompanied by alternative splice variants impacting their amino end sequences. The situation is slightly different in the mouse rhomboid system where 9 of the 13 genes contain variants impacting the amino end. Based on the mouse database, Rhbdf2, Rhbdd1 and Rhbdd3 do not so far have any variants impacting this region. Interestingly, only 3 Arabidopsis rhomboid genes have entries with predicted alternative splicing within the amino region - RBL4, RBL14 and RBL15. One Drosophila gene, Stet, has an altered amino terminus resulting in an alternative start methionine. Of the 5 C. elegans genes, one has an altered amino terminus due to alternative splicing. This gene, ROM-4, displays a frame-shift that shortens the length of the region.\n\nChanges to the L1 Loop region – The L1 region contains a conserved “loop” structure. This structure lies either side of transmembrane helices and plays a role in rhomboid protease activity32. The L1 loop is also partially inserted into the membrane54. Site-directed mutagenesis of the conserved L1 loop residues revealed evidence that this loop controls the way in which rhomboids interact with lipid membranes54. Our analyses of the Figure 1 entries indicate that the L1 Loop region is likely subject to alternative splicing (Figure 2–Figure 6 and Table S5).\n\nIn human, splice variants involving the L1 loop accompany 9 different rhomboid genes. Generally, the outcomes of the variants range from deletion of residues from part of the L1 loop region, to altering a few residues at one end or the other of the structure.\n\nA similar trend was found for 4 of the mouse rhomboid genes, Rhbdf1 (iRhom1), Rhbdd2, Derlin2, and Ubac2. Again, the two splice events resulted in the loss of residues from the L1 loop.\n\nThe Arabidopsis database contains splice variants of the L1 loop for two genes, RBL4 and RBL14. Like in human and mouse, alternative splicing resulted in the removal of residues or large sections of the L1 loop.\n\nThe C. elegans ROM-4 gene exhibits an alternate start methionine and a frame-shift within the L1 loop, giving rise to only the beginning part of the loop.\n\nChanges to regions affecting other structural aspects - This category is defined as changes to the linker or other transmembrane regions (TMD) with no currently assigned functions, as opposed to the regions with distinct functions discussed above and below. Since changes to these regions, subtle or extensive, could potentially affect the other functional aspects of the protein itself, or its interactions, it would be important to assess these splicing events.\n\nIn human, 11 of the 13 rhomboid gene entries examined are accompanied by splice variants in regions of the protein that fall under this category. Examples being PARL variants lacking TMD3 and part of TMD4, or the amino end of TMD1 (see Figure 3–Figure 6 and Table S9).\n\nChanges to neighbouring regions of the catalytic dyad - The catalytic sites of rhomboid proteases consist of residues contributed from two different transmembrane domains, TMD4 and TMD6, when using the “6+1” model1. PARL and secretase-type catalytic residues are characterized by the amino acids GxSx – H. The catalytic residues of iRhoms are characterized by the residues GPxx – H.\n\nThere are currently 4 entries for human PARL rhomboids accompanied by splice variants that impact catalytic potential by altering the GASG or H sites through limited deletions, or extensions and the subsequent loss of the serine and glycine residues (GASG to GA) (Figure 2–Figure 6, Table S6).\n\nBoth human iRhoms, RHBDF1 and RHBDF2 (iRhom1 and iRhom2), contain frame shifts and early terminations within the L1 loop. RHBDL1 has a predicted variant resulting from a frame shift early in the transcript. The RHBDL1 frame shift alters the peptide sequence and removes the catalytic residues. The predicted mRNA/peptide for RHBDL3 displayed the same outcome as RHBDL1. The RHBDD1 gene is accompanied by two predicted forms with frame shifts and early terminations occurring before the catalytic residues.\n\nIn mice, there are 3 entries in our data set with splice variants that impact the catalytic residues. Rhbdf1 (iRhom1) is associated with extensive alternative splicing predictions. Nine different forms are predicted to impact the catalytic potential of the protein. Eight of these predictions display 2 additional residues within TMD4 (GPAG catalytic site), a loss of a residue within TMD6, and a changing of the histidine to a proline. The predicted transcript for the ninth form contains a frame shift in TMD2 that ultimately impacts the catalytic sites. Rhbdf2 has a predicted form with a frame shift in TMD2. Rhbdl3 gene is also accompanied by a splice variant with the potential to alter catalysis. Four of the seven forms result in a frame shift within TMD3 with resulting alterations to the catalytic regions. The resulting peptides remain out of frame, altering the peptide sequence downstream from TMD3.\n\nIn our Arabidopsis entries, 3 genes show evidence of altered catalytic potential through alternative splicing. One At1g74130 mRNA database entry exhibits a frame shift and early termination. The early termination resulted in the removal of the last TMD which basically eliminates the final catalytic residue. Two additional splice variants of At1g74130 were discovered experimentally by Powles et al.47. These two variants displayed similar outcomes as the predicted form from the database entry above, namely early termination sites resulting in two different lengths at the carboxyl end of the protein. RBL3 (At5g07250) is accompanied by a form where the TMD containing the catalytic histidine is removed entirely. Although the Gate and the catalytic histidine residue are removed, the predicted carboxyl terminus of RBL3 is maintained in this RBL3 variant. The last Arabidopsis gene to highlight in this category is RBL 14 (At3g17611). RBL14 is accompanied by a splice variant that may alter the catalytic potential of this rhomboid by using an alternate start methionine located immediately after the catalytic GFSG residues.\n\nC. elegans ROM-4 has an alternate starting methionine and a frame shift that results in the removal of the catalytic residues.\n\nChanges to neighbouring regions of the L5 Cap and the transmembrane domain 5 Gate (TMD5) – Based on the 6+1 rhomboid protein model1, transmembrane domain 5 (TMD5) is postulated to be a feature that controls the entry of a substrate into the active site - a gating control that determines enzymatic activity55,56. The Gate (TMD5) appears to be a region of potentially active alternative splicing activity (Figure 2–Figure 6, Table S7).\n\nOf the 13 human rhomboid genes with entries in our data set, 6 are accompanied by alterations to the Gating TMD. PARL, RHBDF1, RHBDL1, RHBDL3, DERL2 and DERL3 are accompanied by forms with altered TMD6 (based on the 1+6 model). The most common resulting event appears to be early termination of the protein.\n\nThe same splicing outcomes appear to be present in our set of entries for mouse, Arabidopsis and C. elegans rhomboids. Mouse Rhbdl3, Rhbdf1 and Rhbdf2 (iRhom1 and iRhom2) are accompanied by variants with deletions or early terminations caused by frame shifts. The Arabidopsis data set contains two genes with predicted alternative spliced sequences affecting the gating TMD region. Arabidopsis RBL1 possesses an insertion between the catalytic TMD4 and the linker to the Gate. The RBL3 variant is missing both the gating TMD5 and the catalytic TMD6. The C. elegans ROM-4 variant is also missing the gating TMD as a result of a frame shift.\n\nChanges to the carboxyl terminus region - Most of the carboxyl termini changes are due to frame shifts, giving rise to different carboxyl sequences. In human, PARL, RHBDF2, RHBDL1, RHBDD1, and Derlin3, all contain variants with different carboxyl ends. The situation is similar in mice where Rhbdf1, Rhbdf2 and Rhbdl3 are accompanied by variants with different carboxyl ends (Figure 2–Figure 6, Table S8).\n\nIn Arabidopsis, At1g74130 and At5g07250 variants have shortened carboxyl termini. The three alternative splice variants of At1g74130 lack the entire predicted carboxyl terminus. The introduction of a stop codon in TMD6 (or further upstream) resulted in the early termination of translation. The At5g07250 (RBL3) variant lacks TMD5 and TMD6, but the carboxyl terminus is restored with the removal of the first 4 residues of the predicted motif.\n\nChanges to the 3’ UTR region of the transcript - There are also changes associated with 3’ UTR of rhomboid transcripts (Table S10). In our set of entries, there are 9 human genes with splice variants in the 3’ UTR. Some of the variants possess longer 3’ UTR sequences, whereas others exhibit shorter 3’ UTRs.\n\nA similar situation is observed for mice where 5 genes are associated with variants containing altered 3’ UTRs. Rhbdl2, Rhbdf1 and Rhbdf2 variants contain extended 3’ UTRs, whereas Rhbdl3 variants exhibit shorter 3’ UTRs.\n\nThe Arabidopsis genes At1g74130, At3g17611 and At3g58460 are accompanied by one variant each with shortened 3' UTRs. One other gene, At2g29050 (NM_001084504.1), is represented without a predicted 3' UTR in the database.\n\nThe data compiled above suggest potentially impactful changes to the functionality of the affected proteins, but this speculation is limited to linear protein sequences and motifs. We were next interested in testing out possible functionality changes using currently available predictive tools for 3-D protein structures, despite this highly speculative assessment tool. To this end, we decided to use the established 3-D structure/model of the bacterial rhomboid GlpG to test the potential impact exerted by the various splice variant types. The GlpG model is the most established of the rhomboids and offers a more complete structure for this analysis. It should be noted that there are caveats associated with the use of the bacterial GlpG to assess other rhomboid types, but this analysis is strictly focused on how changes could theoretically impact such a rhomboid structure. Because these assessments are judged as being too speculative, the outcomes are provided only as Supplementary Material (Supplementary File 1 and Figure S1–Figure S8). These outcomes may be useful starting points for guiding future structural studies that validate the outcomes. The actual impacts to functionality and structure of particular alternatively spliced protein variants also need to be studied individually through experimentation. This notion was assessed here for a selection of splice variants using the different types of activity tests. These tests were devised mainly to uncover evidence of functionality in splice variant proteins, as opposed to assigning possible biological roles. Different contexts were used to assess functionality of the selected splice variants, contexts ranging from transgenic expression to assays using recombinant proteins as exogenous additives.\n\nThe first series of tests were conducted for the Arabidopsis At1g74130 splice variants to obtain verification of functionality in a heterologous transgenic setting. For At1g74130, a functional relationship between its splice variants and a known yeast mitochondrial rhomboid substrate, Mgm1, was initially discovered using transgenic yeast47. As shown previously in Powles et al.47, each At1g74130 splice variant impacted the Mgm1 ratio (the amount of uncleaved (97 kDa) to cleaved (84 kDa)). The At1g74130 M and S splice variants individually reduced the ratio by about a third (from ratios of 0.67–0.7 to 0.45 for M and 0.39 for S)47. Since the At1g74130 splice variants exist simultaneously in their natural Arabidopsis context, we further assessed different combinations of the same splice variants to see if such combined interactions influence the Mgm1 ratio, an indicator of splice variant activity. Further adjustments to the ratios by the various combinations of splice variants would uncover additional evidence of interaction and functionality. The results in Figure 7D (Dataset 157) indicate that various variant combinations possess the ability to influence the Mgm1 ratio, mainly more uncleaved Mgm1 (triggering higher ratios, from 0.81–1.09, instead of the ratios observed for cells expressing one variant at a time). The pACT2 control mitochondria displayed a similar Mgm1 ratio to that observed in Powles et al.47, at 0.66 ±0.002 versus 0.70 ±0.002, respectively (Figure 7D and Dataset 157). Yeast cells expressing the pair At1g74130 (L) and (S) displayed a ratio of 1.05 ±0.02, cells expressing At1g74130 (L) and (M) exhibited a ratio of 0.81 ±0.004, and cells expressing At1g74130 (S) and (M) resulted in a ratio of 1.09 ±0.03 (Figure 7D and Dataset 157). The top set of ratios is provided and adapted from Powles et al.47 to contrast the differences between single variant and double variant expression. These results demonstrate that the splice variants exhibit functionality in this setting.\n\n(A) Expression of At1g74130 splice variant proteins in yeast shows activity as increased nystatin sensitivity in a disk assay. Nystatin infused disks were applied on top of transgenic S. cerevisiae lines prepared as top agar cultures at a density of 5 × 107 cells per mL. The transgenic yeast line being assessed is indicated: The pACT2 control plasmid line (top left), the line expressing the At1g74130 (L) variant (top right), the line expressing the At1g74130 (S) variant (bottom left) and the line expressing the At1g74130 (M) variant (bottom right). A representative experiment is presented in this panel. (B) The areas of clearance (degree of cell death) around the nystatin infused disks were compared for the above set of plates (panel A). The pACT2 control line is labelled as pACT2 Control. The splice variant-expressing lines are labelled as in panel A. The average areas of clearance are shown with error bars (standard deviation) (n=10). (C) Expression of At1g74130 variants in yeast also shows activity as changes to susceptibility to nystatin and amphotericin B. The same lines used in panels A and B were tested here. Each line was treated and then plated on appropriate agar media to determine susceptibility. The Percent Survival was calculated relative to untreated condition (untreated or mock treatments with no antifungals). The blue bars represent the control (no nystatin or amphotericin B), red bars represent cells treated with amphotericin B (AmB), green bars represent cells treated with nystatin (Nys) and the purple bars represent cells treated with both AmB and Nys. Error bars represent variation between two experiments. (D) Analysis of possible interactions of between yeast Mgm1 and different combinations of At1g74130 splice variant proteins. The top row represents the Mgm1 levels in strains containing pACT2 or expressing one of the At1g74130 variant proteins (note that these immunoblotting results were reported previously from Powles et al.47 and adapted here for comparative purposes). The bottom row of immunoblots depicts the changes in the ratio of Mgm1 (the uncleaved 97 kDa form versus the cleaved 84 kDa form) when various combinations of At1g74130 splice variant proteins were co-expressed. Each representative immunoblot is labelled accordingly. Only the relevant parts of the immunoblot images are displayed. The ratios were derived from two experiments and the bars represent variation between the two experiments. The immunoblot images used for analyzing the rhomboid combination experiments are provided in Figure S9.\n\nWe next assessed activity by looking at changes in sensitivity to the fungicide nystatin. Changes to sensitivity was assessed in two ways, growth/survival of yeast cells around nystatin-infused disks as a longer treatment strategy and nystatin treatments of cell cultures as a transient strategy. As shown in Figure 7A and B (Dataset 157), the expression of At1g74130 variants in yeast (especially (S) and (M)) increased nystatin sensitivity and cell death. The pACT2 control yeast strain and the strain expressing At1g74130 (L) displayed similar average areas of clearance (between 40–50 mm2), whereas the strains expressing At1g74130 (S) and (M) frequently displayed larger clearance areas of approximately 60–70 mm2 on average.\n\nThe disk assay results were reflected in the liquid culture assays. Yeasts expressing the same At1g74130 variants were treated transiently with nystatin and amphotericin B prior to plating. Compared to untreated cells, all strains experienced decreased survivability when treated with nystatin and/or amphotericin B (Figure 7C and Dataset 157). All cells displayed decreased survival when treated with amphotericin B. The pACT2 control was around 60.13 ±2.83% and the splice variant lines were around 74–76% (At1g74130 (L) line was at 75.91 ±0.83%, (S) line was at 75.49 ±5.99% and (M) line was at 74.26 ±2.04%). When treated with nystatin, all At1g74130 lines displayed decreased survivability relative to the control strain (pACT2 control line was at 30.82 ±4.85% compared to At1g74130 (L) line at 15.98 ±1.48%, (S) line at 15.07 ±0.77%, and (M) line at 19.52 ±6.25%). When treated with both amphotericin B and nystatin, the control line displayed 15.19 ±0.45% survival, whereas the splice variant lines were in the 6–12% range (At1g74130 (L) line at 6.62 ±1.20%, (S) line at 11.61 ±3.33%, and (M) line at 8.39 ±1.90%). Overall, with the exception of the amphotericin B alone treatment, cells expressing any of the three At1g74130 splice variants displayed lower survivability when treated transiently with fungicide. Both of the fungicide settings (panels A and B; Dataset 157, and then panel C; Dataset 157) indicate that the At1g74130 splice variants possess functionality. The phenomenon observed in transgenic yeast were also reflected in the transgenic bacteria setting, where ß-lactamase expression and secretion are high and considered to represent a “Superbug” (antibiotic resistance) model. Enhanced sensitivity to ampicillin was observed in bacteria expressing the At1g74130 (L) and (S) splice variants relative to (M) (Figures 8A and B; Dataset 258, respectively). Bacteria expressing At1g74130 (L) and (S) exhibited higher levels of sensitivity at lower ampicillin concentrations relative to (M) in this context. At the protein level, bacteria expressing At1g74130 splice variants displayed reduced synthesis, processing and secretion of β-lactamase (Figure 8C). In cells with pET20b only, most of the ß-lactamase were present in the mature form (29 kDa) and at high total cell levels. In contrast, bacteria expressing At1g74130 splice variants exhibited shifts toward the precursor form (31.5 kDa), with (L) being the most impacted (Figure 8C). Additionally, there were lower levels of β-lactamase overall in these same cells that may further contribute to the higher levels of sensitivity (Figure 8C). These results indicate that the splice variants display functionality in this setting.\n\n(A) Bacterial cells expressing one of the three different splice variant proteins were grown on LB-ampicillin plates from 1.0 mg/mL to 1.75 mg/mL (labelled L, S, and M, as in the previous figures). (B) Graphical representation of the survival levels observed for each of the plates shown in panel A (reflected as the numbers of colonies growing). The labelling is the same as in panel A. (C) Expression of At1g74130 variant proteins in bacteria alters production and secretion of β-lactamase. Immunoblot images of β-lactamase protein samples are shown for different bacterial strains grown in different ampicillin concentrations. The lanes contain lysates from control cells containing pET20b only (labelled Control), and one of the At1g74130 variants, L, S, and M (labelled as Variant L, S, or M). The smaller-sized bands marked “M” represents the mature β-lactamase form (29 kDa) and the larger-sized bands marked “P” represents the precursor β-lactamase form (31.5 kDa). The full immunoblot image used is provided in Figure S10. (D) Recombinant At1g74130 variant proteins were tested for activity (enhanced sensitivity to ampicillin in this case) as exogenous additives in the same manner as that shown for yeast in Figure 7. Cells (resulting colonies) surviving the different treatments are depicted as Percent Survival in the graph. The error bars represent standard deviations (n=3). The Percent Survival was calculated relative to the No Treatment control cell numbers. “No treatment” represents the bacterial culture used (diluted to the prescribed cell number tested as described in Methods). The variant protein being tested is labelled as in the panel A. DMSO indicates the use of DMSO (5% v/v) as the delivery agent. The Mock Treatment contains all components used except with no recombinant proteins added. All treatments involve exposure to 1.25 mg/mL ampicillin.\n\nEvidence of functionality was also observed using an exogenous additive approach, where recombinant splice variant proteins were used to pre-treat cells before testing for changes to antimicrobial sensitivity (see Methods). This treatment scheme would be considered a transient strategy. For yeast cells, recombinant splice variants were delivered using a sub-lethal level of amphotericin B as the pre-treatment step before testing for sensitivity (survival) to nystatin. Even though amphotericin B is a fungicide (especially at higher levels), it was feasible to utilize amphotericin B at sub-lethal levels (1% (v/v)) for delivery purposes because this compound is capable of altering the permeability of fungal membranes and allow rhomboid proteins cellular access. The treatment matrix used for these yeast assays is shown in Figure 9A. The yeast strain tested here was the same parental host line used in the earlier assays. Sensitivity to nystatin was then tested at a level of 0.5% (v/v). Overall, treatment resulted in smaller colonies at the time of plate growth documentation (compare treatment 1 to treatments 2 to 9 in Figure 9B and C; Dataset 359). All control or mock-type treatments display higher survival percentages compared to the three recombinant rhomboid pre-treatments (Figures 9B and C; Dataset 359). Relative to the Positive Control (considered 100% survival), the amphotericin B only treatment (AmB Control) displayed 96.46 ±0.43% survival, nystatin only (Nys Control) showed 85.46 ±2.60%, the amphotericin B-nystatin treatment (AmB/Nys Control) was at 81.81 ±1.86%, the amphotericin B-nystatin with BSA (BSA Control) was at 88.88 ±2.24% and amphotericin B-nystatin with protein elution buffer (Elution Control) was at 83.23 ±2.53%. Other components of the fungicides, such as the deoxycholate present in the amphotericin B solution, were also tested and found to have no impact on survivability at the levels used in these assays (Figure S11). Finally, all three treatments with recombinant splice variants (which includes amphotericin B and nystatin) displayed decreased survivability (albeit at different levels between 6–14%) relative to control treatments (pre-treated with At1g74130 (L) displayed 13.93 ±1.16% survival, (S) at 6.92 ±4.39%, and (M) at 10.08 ±2.53%). Yeast cells pre-treated with recombinant At1g74130 variant proteins and then treated with both amphotericin B-nystatin exhibited the smallest colony sizes (Figure 9B). Only pre-treatments with recombinant splice variants and amphotericin B resulted in higher sensitivity to nystatin. Protein pre-treatments without the delivery agent amphotericin B behaved like the controls (Figure S11).\n\n(A) The typical treatment matrix used in this study is shown. The check marks in the columns indicate the presence of a treatment component. The variant protein tested in Figure 7 is labelled only as L, S, and M in the matrix. The final treatment volume in all cases was 100 µL. (B) The corresponding plate is shown for each of the treatments depicted in panel A (labelled according to treatment numbers 1 to 9). (C) Cells (resulting colonies) surviving the different treatments are depicted as Percent Survival in the graph. The treatments are in the same order as presented in panels A and B. The error bars represent standard deviations (minimally n=3).\n\nChanges to ampicillin sensitivity in bacteria were also observed using the transient approach. As a commonly used delivery component in many drug applications, DMSO was utilized here as the protein delivery agent in place of amphotericin B. Bacteria were pre-treated with DMSO and recombinant proteins before testing ampicillin sensitivity. Relative to the untreated (media only) and mock treatment (all components without recombinant proteins), pre-treatments with exogenously added At1g74130 splice variants decreased the number of colonies (a proxy for cells surviving the treatment) at the time of plate growth documentation (Figure 8D and Dataset 258). The mock treatment did not differ significantly (T-test, P = 0.36) from the no treatment control, indicating that the components used in the buffer did not contribute significantly to the enhanced level of ampicillin sensitivity. Relative to the mock treatment or no treatment control, the pre-treatment of bacteria with recombinant protein additives At1g74130 (L), (M), or (S), exhibited significant reductions in colony numbers (T-test: P = 0.022, P = 0.026, P = 0.029, respectively). The No Treatment control using 1.25 mg/ml ampicillin without DMSO, represents the reference point of 100% survival. The Mock Treatment without protein additives and with DMSO resulted in 95.85 ± 6.08% survival. The treatments (5% DMSO and 1.25 mg/ml ampicillin) and pre-treated with At1g74130 (L), (M), or (S) resulted in 71.36 ± 8.96%, 79.54 ± 3.10%, and 77.00 ± 1.27% survival, respectively.\n\nThe functionality assays used for At1g74130 were applied to splice variants from two other categories of rhomboid proteins. One splice variant pair was derived from Arabidopsis At1g25290 (named (L) and (S)) and another variant originated from human Ubac2. At1g25290 was from the “Active Rhomboid Proteases” category and Ubac2 is from the “Other Inactive Rhomboid Proteins” category. The overall results for variants from these two other categories indicate functionality as well in our assay settings (select assay results are reported here).\n\nFor the At1g25290 splice variants (L) and (S), similar responses were observed in the exogenous additive-transient setting, albeit with differences in impact from that observed for the At1g74130 variants. Functionality was displayed in both bacterial (Figure 10A and Dataset 460) or yeast cell settings (Figure 10B and Dataset 460). The outcomes between the two At1g25290 splice variants were themselves different. The different sensitivity levels displayed by (S) relative to (L) suggest that the phenomenon observed is attributed to the added recombinant rhomboid variant (that their differences were derived from alternative splicing) and not to other components in the mixtures.\n\n(A) Recombinant At1g25290 splice variant proteins L and S were tested for activity (for enhanced sensitivity to ampicillin in this case) as exogenous additives and bacteria. The assays were conducted in the same manner as that shown in Figure 8 (from untreated to mock to added proteins). Cells (resulting colonies) surviving the different treatments were then assessed and represented as Percent Survival. The key results comparing the splice variants L and S are shown in this panel. The bar graphs are arranged to the corresponding representative results, the resulting agar plates. The error bars represent standard deviations (n=4, T-test, P=0.01). (B) Recombinant At1g25290 splice variant proteins L and S were also tested for activity (for enhanced sensitivity to nystatin in this case) as exogenous additives and yeast. The assays were conducted in the same manner as that shown in Figure 9 (from untreated to mock to added proteins). The organization of panel B is the same as in panel A, except for yeast and nystatin. The error bars represent standard deviations (n=4, T-test, P=0.01).\n\nThe human Ubac2 splice variant tested is a fusion between a rhomboid protein sequence (considered a pseudoprotease) and ubiquitin-associating domains61,62. Like the above phenomenon, recombinant Ubac2 variant proteins showed functionality as an additive in bacterial and yeast assays, albeit at a more modest level of influence on antimicrobial sensitivity (Figures 11; Dataset 563).\n\n(A) Recombinant splice variant proteins At1g25290 (L) and one Ubac2 were tested for activity (for enhanced sensitivity to ampicillin in this panel) as exogenous additives and bacteria. The assays were conducted in the same manner as that shown in Figure 8 (from untreated to mock to added proteins). Cells (resulting colonies) surviving the different treatments were then assessed and represented as Percent Survival. The key results comparing the splice variants At1g25290 (L) and Ubac2 are shown in this panel. The error bars represent standard deviations (n=4, T-test, P=0.1). (B) Recombinant At1g25290 splice variant protein L and Ubac2 were also tested for activity (for enhanced sensitivity to nystatin in this panel) as exogenous additives and yeast. The assays were conducted in the same manner as that shown in Figure 9 (from untreated to mock to added proteins). The organization of panel B of this figure is the same as in its panel A, except for yeast and nystatin. The error bars represent standard deviations (n=4, T-test, P=0.1).\n\nIn whole, not all variants will show functionality in the different test settings. The different assays does however provide evidence that splice variants from different rhomboid categories possess functionality, supporting the notion derived from the in silico analysis, that alternative splicing provides a mechanism for diversifying the numbers of working rhomboid proteins, and the roles of rhomboid proteins play in a particular system.\n\n\nDiscussion and conclusions\n\nAlternative splicing is used by many organisms to control and to diversify protein function. Historical examples include human tropomyosin, human kallikreins (secreted serine proteases), and fungal Ski7/Hbs proteins64–66. The same appears to be possibly happening with rhomboid genes, but despite witnessing alternative splicing as a mechanism for diversifying functionality in Arabidopsis and human breast cancer cells35,41,45–47,51,67, the number of demonstrated cases remains limited. We were thus interested in assessing how often this happens using the information available in the different genetic databases. Our periodic database analysis over a five year period was focused specifically on functionality, as opposed to function, to capture slight changes (potential changes at this juncture) to functionality, such as those reported above46,47. We also wanted to determine the extent of alternative splicing within the different rhomboid gene systems of a particular organism as well as between different species. We thus limited our analysis only to splice variant entries in current RNA sequence databases of select model organisms with completely sequenced genomes. It was important to limit our analysis so that we can address the issue of extent appropriately and then assess how alternative splicing is used to modify the functionality of distinct regions of the affected rhomboid proteins of our data set, especially regions with defined purposes. We then assessed the overall notion of the in silico findings by testing a selection of variant proteins from three different categories.\n\nThe overall in silico evidence supports the hypothesis that alternative splicing is likely used to diversify rhomboid functionality in a number of cases. This is especially the situation in human, mouse, and Arabidopsis, organisms with relatively high numbers of rhomboid or rhomboid-like genes. Currently, there is a total of 95 entries for 13 human rhomboid genes that reflect alternative splice products, 53 for 13 mouse genes and 40 for 22 Arabidopsis genes. These splice variants were also not limited to a particular rhomboid category. The diversification appears to occur generally in distinct groupings across the entire rhomboid protein sequence. Although the in silico data suggest potentially impactful changes to functionality, this speculation remains limited to linear protein sequences and motifs. Therefore, we next tested the possible changes to functionality using currently available predictive tools for 3-D protein structures, despite the highly speculative nature of these tools. This analysis was focused strictly on how changes could theoretically impact such a rhomboid structure. Because these assessments are judged as being too speculative, the outcomes of these tests are provided only as Supplementary Material (Supplementary File 1 and Figure S1–Figure S8). These outcomes may be useful for guiding future structural studies as well as validating the outcomes through extensive experimentation. The notion of diversification in functionality by alternative splicing mechanisms was tested experimentally using recombinant proteins of six different splice rhomboid variants from three different categories, Active Rhomboid Proteases, Inactive Rhomboid Proteins, and Other Inactive Rhomboid Proteins. These splice variants represent different structural changes from active sites, to truncations, to fusions.\n\nBased on the compiled in silico data, some of the potential impacts were quite extensive and obvious. Some of the more obvious ones appear to arise from subtle changes to the protein sequence, such as the introduction or removal of a few residues. Many impacted important structural motifs, sometimes from afar or indirectly (Figure 2–Figure 6 and Figure S1–Figure S8). Although the degree of amino acid sequence conservation of rhomboid and rhomboid-like proteins is relatively low between species and types, there are distinct conserved residues/motifs that serve the same important functions. Some of the functional motifs potentially impacted did include motifs of known functions like the L1 loop, the TMD5-L5 cap, and the catalytic dyad region. Such situations are likely to bear significant consequences with respect to functionality. For instance, the L1 loop has been the focus of several studies because it contains one of the conserved motifs outside of the catalytic cluster54. Although the function of the L1 loop is not entirely understood, the importance of the loop on protease functionality was demonstrated by mutagenesis experiments32,54. Normally, the L1 loop is partially embedded in the membrane. Mutation of the conserved WR motif in the L1 loop of GlpG decreased proteolytic activity, suggesting a modulatory role for the loop54. Additional evidence suggests a regulatory role for the L1 loop, and this role is linked to the formation of a rigid L1 loop structure54. If the L1 loop serves as an anchor to the lipid bilayer, alterations to this structure could modulate the protein’s enzymatic activity with substrates. In 2007, Baker and coworkers56 found an enhancement of proteolytic activity with their set of mutagenized L1 loop experiments, which further demonstrates the link between L1 loop and functionality. Such outcomes are certainly possible and were observed in the 3-D models predicted for the different splice variants tested here (Figure S7).\n\nSimilar outcomes were also observed for splice variants involving changes to the L5 cap and TMD5 region (Figure S8). In addition to experiments aimed at the L1 loop, Baker and coworkers56 also carried mutagenesis-based experiments on the TMD5 region and observed enhancement of cleavage activity with some of the structural changes in TMD5. It was hypothesized that destabilization of the TMD5 helix allowed enhanced substrate entry56. TMD5’s destabilization is believed to alter its configuration by changing its angle/tilt and proximity to neighboring helices. This in turn alters the efficiency of gating by this region. Alterations to the efficiency of gating then in turn affects proteolytic activity56. This is because, normally, when the TMD5 is positioned in the 'open' conformation, the TMD5 helix pulls the L5 loop outward55. The movement of the L5 cap structure allows substrate entry into the catalytic cavity. The open conformation of the L5 cap is also believed to permit the entry of water into the catalytic cavity55. Therefore, alterations to TMD5 and the L5 cap could potentially change features that affect substrate entry. The predicted outcomes in the examples shown in Figure S8 could possibly manifest in a similar manner with equally consequential effects. The structural outcomes revealed in our theoretical assessment of splice variants are thus likely to represent a mechanism for diversifying rhomboid functionality, since these alternative splice variants likely exist in the organisms studied.\n\nBased on a combination of the previous findings for the two Arabidopsis plastid rhomboids46,47, the human RHBDD245, and the trends revealed in this study, the overall evidence suggests that alternative splicing is a functionally significant mechanism for diversifying rhomboid functionality. This means that splice variants of rhomboids and rhomboid-like proteins likely exist simultaneously in the cell or sub-cellular compartment. Like the two Arabidopsis plastid proteins, the alternative splice variants are likely co-expressed, modulated relative to each other to respond to the cell’s needs, and interacting in some manner. The possibility of interactions between rhomboid units themselves has been reported by Wu et al.55 for the bacterial rhomboid protease GlpP. Such interactions with different rhomboid variants/forms and populations could therefore manifest in a number of ways that affect rhomboid functionality. The theoretical approach used in this study, and the predicted outcomes that may arise are thus not without merit, and should be considered as guidance for further experimentation. The possibilities, such as the ones discussed in the above examples, are observed experimentally in other studies and in functionality assays conducted for our select splice rhomboid variants. There are a number of other experimentally tested examples where truncations have been observed to impact functionality. In addition to rhomboid proteins, examples of other types of proteins include those recently reported by Stoddart et al.68 for an integral membrane pore, and by Quemeneur et al.69 where shape influences protein mobility within membranes. Whatever the situation may be for rhomboids, it is clear that it is necessary to characterize splice variants for each rhomboid and to determine how splicing influences rhomboid functionality. This would be important for elucidating how the different rhomboids work as a network.\n\n\nData availability\n\nDataset 1: Raw data underlying the results in Figure 7. Figure 7B. Disk Assay Area Measurements of Growth (Sensitivity) in the Presence of Nystatin for Transgenic Yeast Lines Expressing At1g74130 Splice Variants; Figure 7C. Percent Survival (Cell/Colony Counts) of Various Transgenic Yeast Lines When Treated with Nystatin and Amphotericin B; Figure 7D. Densitometry Assessment of Immunoblots Developed for Assessing Mgm1 Levels and Ratios in Mitochondria Isolated from Transgenic Yeast Lines Expressing At1g74130 Splice Variants. 10.5256/f1000research.13383.d19216157\n\nDataset 2: Raw data underlying the results in Figure 8. Figure 8A–B. Cell/Colony Counts of Various Transgenic Bacterial Lines When Grown on Plates with Increasing Ampicillin Concentrations; Figure 8D. Assays to Determine Percent Survival (Cell/Colony Counts) of Bacterial Cells When Treated Exogenously with Recombinant At1g74130 Splice Variant Proteins. 10.5256/f1000research.13383.d19216258\n\nDataset 3: Raw data underlying the results in Figure 9. Figure 9C. Assays to Determine Percent Survival (Cell/Colony Counts) of Yeast Cells When Treated Exogenously with Recombinant At1g74130 Splice Variant Proteins. 10.5256/f1000research.13383.d19216359\n\nDataset 4: Raw data underlying the results in Figure 10. Figure 10A. Assays to Determine Percent Survival (Cell/Colony Counts) of Bacterial Cells When Treated Exogenously with Recombinant At1g25290 Splice Variant Proteins; Figure 10B. Assays to Determine Percent Survival (Cell/Colony Counts) of Yeast Cells When Treated Exogenously with Recombinant At1g25290 Splice Variant Proteins. 10.5256/f1000research.13383.d19216460\n\nDataset 5: Raw data underlying the results in Figure 11. Figure 11A. Assays to Determine Percent Survival (Cell/Colony Counts) of Bacterial Cells When Treated Exogenously with a Recombinant Ubac2 Splice Variant Protein; Figure 11B. Assays to Determine Percent Survival (Cell/Colony Counts) of Yeast Cells When Treated Exogenously with a Recombinant Ubac2 Splice Variant Protein. 10.5256/f1000research.13383.d19216563", "appendix": "Competing interests\n\n\n\nParts of this study may be considered as being potentially related to material contained in a patent application (US 2016/0129078 A1; available here). There are no other competing interests disclosed at the time of submission.\n\n\nGrant information\n\nThe research was supported in part by a grant (number 43698) from the Natural Sciences and Engineering Research Council of Canada (to K.K.) and by funds from Queen’s University (to K.K.). Joshua Powles was also supported by graduate awards from Queen’s University.\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 thank Nicholas Ostan and Casssandra Nelson for their technical assistance in some of the activity assays and data collection (both were at Queen’s University during their roles on this research project).\n\nPart of the work presented in this publication was derived from the doctoral thesis by the first author, Joshua Powles. Dr. Powles’s doctoral thesis was deposited according to graduate program requirements into the Queen’s University electronic repository for graduate theses and dissertations.\n\n\nSupplementary material\n\nSupplementary File 1: Supplementary notes for Figures S1 to S8; References cited in the supplementary notes for Figures S1 to S8; Supplementary figure legends for Figures S1 to S11.\n\nClick here to access the data.\n\nTable S1. List of alternative splice sequence entries sorted by model organisms.\n\nClick here to access the data.\n\nTable S2. List of names and alternate names used for the various rhomboid genes and proteins.\n\nClick here to access the data.\n\nTable S3. Summary of alternative splice variants impacting the 5’ UTR and translation.\n\nClick here to access the data.\n\nTable S4. Summary of alternative splice variants impacting the amino terminus.\n\nClick here to access the data.\n\nTable S5. Summary of alternative splice variants impacting the L1 loop region.\n\nClick here to access the data.\n\nTable S6. Summary of alternative splice variants impacting regions of the catalytic dyad.\n\nClick here to access the data.\n\nTable S7. Summary of alternative splice variants impacting the L5 cap region.\n\nClick here to access the data.\n\nTable S8. Summary of alternative splice variants impacting the carboxyl terminus.\n\nClick here to access the data.\n\nTable S9. Summary of alternative splice variants impacting the other regions and motifs.\n\nClick here to access the data.\n\nTable S10. Summary of alternative splice variants impacting the 3’ UTR.\n\nClick here to access the data.\n\nFigure S1. Examples of alternative splicing and their potential impacts on the structure of the amino terminal region.\n\nClick here to access the data.\n\nFigure S2. Examples of alternative splicing and their potential impacts on the structure of the carboxyl terminal region.\n\nClick here to access the data.\n\nFigure S3. The potential impact of alternative splicing on the structure of the Arabidopsis rhomboid At3g17611.\n\nClick here to access the data.\n\nFigure S4. The potential impact of alternative splicing on the structure of the Arabidopsis rhomboid At3g53780.\n\nClick here to access the data.\n\nFigure S5. Examples of alternative splicing and their potential impacts on the L1 loop structure.\n\nClick here to access the data.\n\nFigure S6. Examples of alternative splicing and their potential impacts on the L5 Cap-TMD5 structure.\n\nClick here to access the data.\n\nFigure S7. The potential impact of alternative splicing on the structure of the Arabidopsis plastid rhomboid At1g25290.\n\nClick here to access the data.\n\nFigure S8. Examples of alternative splicing and their potential impacts on the structure of the catalytic region.\n\nClick here to access the data.\n\nFigure S9. Immunoblot images used for analyzing the impact of different rhomboid combinations on the Mgm1 ratios presented in Figure 7D.\n\nClick here to access the data.\n\nFigure S10. Immunoblot images used for analyzing the impact of rhomboid variants on ß-lactamase production and secretion presented in Figure 9C.\n\nClick here to access the data.\n\nFigure S11. Assay testing the combination of \"Inactive\" plastid rhomboid protein variants and deoxycholate in yeast without amphotericin B.\n\nClick here to access the data.\n\n\nReferences\n\nLemberg MK, Freeman M: Functional and evolutionary implications of enhanced genomic analysis of rhomboid intramembrane proteases. Genome Res. 2007; 17(11): 1634–1646. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "30490", "date": "22 Feb 2018", "name": "Martin Kollmar", "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\nJoshua Powles and Kenton Ko conducted a study on rhomboid serine proteases with respect to their alternative splice variants. The study is based on a discovery of the same researchers in 2012 that two of the Arabidopsis thaliana homologs contain alternative splice variants. The manuscript displays several conceptual problems that would demand a major revision if touched. Therefore I suggest the authors to completely revise all in silico analyses and completely re-write the paper. The problems are as follows:\n=> The authors provide much unneeded information for certain aspects (which would just be a style problem) but, most importantly, miss a lot of essential information to recapitulate and redo their analyses. Example: The authors state in the Methods section: “All variant RNA sequences were assumed to result in the generation of functional proteins, including extensively truncated products.” This, however, is not a method. A method would be that the authors write, that they used tool-x or tool-y to translate the RNA sequences, which they subsequently analysed by tool-z. Instead, the authors continue: “The resulting predictions did not indicate that this assumption would not hold true.” What does “resulting” mean here? Which tool was used? A translation of an RNA is usually not termed “prediction”. Which tool was used for the verification? Apart from these style problems, the authors did not provide any evidence that the translations “do” result in functional proteins. It still is just their assumption. In terms of missing information to recapitulate/redo the analyses, the authors do not provide any information on: A) how the set of starting proteins were assembled (search by name? Using BLAST?), B) how the entries were assessed for alternative splicing (on RNA? On protein level? By alignment?), C) the authors claim that their analyses required comparisons between genomic sequences but do not provide any genomic sequence in the main text or supplements, D) the authors claim “alignments with current models of rhomboid proteins” without providing the tool used for doing these alignments nor providing a list or reference for the “current models of rhomboid proteins”, E) the authors state that they used Ni-NTA chromatography for protein purification without providing any buffer. While column washing-buffers likely do not have any effect, the elution buffer has. Was the elution done in a gradient? By a step-wise process? Just by eluting with 500 mM Imidazole? Which pH? Was the purified protein dialysed afterwards? This is essential information, because the proteins were subsequently used in other assays, and these might be compromised by pH or imidazole concentration. F) the authors do not provide any information on ampicillin-concentration used for selection, nor any cell lysis procedure. G) was the purified protein always used directly? Frozen and reused later? How long is the protein stable in which buffer (pH? Imidazole?)? H) I do not understand this sentence: “If used, the rabbit polyclonal anti-rhomboid protein (At1g74130) antibodies were established by our lab and validated as reported in Powles et al. 51 .” Did the authors use this antibody for the western blots? Why did they “validate” the antibody again? I) which media were used for yeast cell growth? Just writing “Cells were grown in glucose-supplemented media” does not make the experiment reproducible. Which glucose concentration? What is a “complete medium”? J) The authors experimentally analyse three variants for the At1g74130 gene, termed L, M, and S, but it is not described anywhere to which splicing events these transcripts belong. Every study that I know that deals with alternative splicing contains a gene structure scheme and shows which exons would be present in which transcript. This is just an incomplete list of examples, where absolutely essential information is missing. The authors would need to check every sentence in the Methods section to see, whether it is really describing a method, and they need to check where and which information is missing.\n=> I have looked at the provided accession numbers for the genes/proteins. The numbers for human and mouse all represent “predicted transcripts”, thus these could all be wrong. I recommend the authors to read a few papers about gene prediction software and pipelines, they will notice that on average every prediction in e.g. human, Drosophila, C.elegans contains 1 wrong exon (e.g. the paper from the eGASP comparison of gene prediction software on these 3 species would be a good starter). Prediction of alternative splice variants is even more error-prone. Thus, the authors should only use transcripts with cDNA evidence in their study (and keep in mind, that cDNA/EST data also sometimes contains errors from e.g. missplicing). Discussing just gene predictions is complete artificial and fiction. The authors could, for example, download a few RNA-seq datasets from the ENCODE and modENCODE data and do the RNA-seq mapping themselves to validate the gene predictions. If the authors cannot do this, they should only analyse and discuss transcript variants for which they find cDNA/EST evidence (and of course the accession numberss for these cDNA sequences need to be given). If the authors did a RNA-seq mapping themselves they could also provide some measure for the likeliness that a suggested variant is a true variant or the result of missplicing, transcription errors, etc. E.g. if thousand of reads are found for a certain gene, is it likely that a variant is a true variant and functional if only supported by 1-2 reads? Or are these 1-2 reads rather representing missplicing and other errors?\n=> The authors claimed several times that they re-did the analysis every 6 months since 2012. Wouldn’t it be a much better way to just look first whether updates on these species were made available at all before spending time in redoing an analysis? I know that these updates only happen occasionally, and not even every second year. Also, the techniques completely changed since about 2010. At least I am not aware of any major study providing new EST/cDNA datasets for the species studied here. All the new data since about 2008 is generated as RNA-seq data. Thus, which further cDNA evidence did the authors expect for the transcripts since 2012 so that they decided to redo the analysis every 6 month? I highly recommend the authors to read all the README-files for the various gene prediction datasets that GenBank and the other databases provide. E.g. Ensemble decided in 2012 (if I remember correctly) to not use any cDNA data anymore for validating their gene predictions. Thus, many exons with cDNA data available (e.g. from isolating and sequencing single genes by research groups) are not present in Ensemble’s gene predictions anymore, if these exons are not predicted by gene prediction tools. Similarly, there are many predicted exons for which no evidence (cDNA/RNA-seq) is available. Please check the GETx-project: There you can see how many RNA-seq reads in each tissue are found for each transcript. You will find out, that for many of the “alternative” transcripts, there is not a single read. The presented discussion of the variants (figures 3 to 6) supports my assumption that most predictions are just wrong. Could the authors provide any reference that it would be possible for such a seven-transmembrane-protein (the rhomboid proteases) to result in a functional protein if e.g. 1 transmembrane region in the middle would be missing due to alternative splicing? If 1 transmembrane region would be missing (this is suggested by several variants the authors discuss) this would turn the direction of the rest of the transmembrane helices and regions: what was inside before would be outside, and what was outside would be inside. To all what I know from membrane protein structures, the transmembrane helices stick together forming a dedicated tertiary structure. Could the authors provide a reference that transcripts with early terminations would ever result in stable and functional proteins, if e.g. only some of the 7 transmembrane helices are present anymore? The C.elegans ROM-4 was stated to contain a variant with just the first TM-helix present. Do the authors really think that this would result in a functional protein? How do the authors exclude that such misspliced/unstable variants would not result in NMD?\n=> The authors claim several times throughout the entire manuscript that they analysed selected model species without giving any information how these were selected. E.g. wikipedia contains a list of about 100 model organisms. Which was the rationale to just look at the 6 in the manuscript? Why not more plants? In this respect, there are many statements of the authors that are just wrong, such as “Currently, of the sequenced model genomes available,”. Of the 100 model organisms at wikipedia (and there are likely more model organisms if other researchers were asked) at least for 80 of them complete genomes are available. Altogether, there are about 5000 eukaryotes with genome assemblies available, of which at least 4000 are complete (e.g. check www.diark.org). In this respect, all the speculations and discussion about which organism contains the most homologs or the most variants are just speculations and should explicitly termed as such. E.g. Brassica species underwent another whole-genome-duplication after separation from the Arabidopsis, and will thus contain many more than the 22 Arabidopsis homologs. Fish also underwent another 1 or 2 (depending on lineage) whole-genome-duplication, therefore will also contain more homologs.\n=> The authors state several times that, although not observed, they expect more alternative variants for the Arabidopsis homologs, because many more variants were identified for human. What is the basis for this expectation? Is there any reference that demonstrates, that Arabidopsis genes have as many alternative splice variants as mammalian genes, on average? All what I am aware of just contradicts this expectation. Arabidopsis genes have fewer exons (thus less possibilities for alternative splicing) and less alternative splice events. Why do the authors not expect more alternative variants for Drosophila or C.elegans, based on their rationale? Shouldn’t yeast have at least some variants (doesn’t have any yet)? Of course, yeast doesn’t, but this should make the authors aware of the problems in their argumentation.\n=> Although the manuscript deals with alternative splice variants, I did not find any reference on the ample literature on this subject. Not even references to a few reviews. Similarly, there is not even mentioning of the accepted types of alternative splicing, e.g. differentially included exons, alternative 5’/3’ splice sites, mutually exclusive splicing, etc. Categorizing the variants detected with these categories would be much more informative than categorizing by region. Based on the descriptions in the supplementary tables, many variants are highly likely just the result from sequencing errors leading to frame-shifts or alternative amino acids. There is only a single alternative splice form that would lead to alternative amino acids for a certain regions, which is mutually exclusive splicing, but this information I did not find anywhere. This could easily be confirmed.\n=> I did not see any gene structure in the manuscript or supplements. The usual procedure in the field is to provide a gene structure drawing of each gene and mark the splicing events on these structures. This is common practice since >30 years. The authors do not show any protein sequence, nor any cDNA sequence, nor any alignment. I cannot see any use of the provided tables in the supplements for other researchers. Terms such as “Original UTR missing, new UTR generated from coding region” (table S3) are not useful. How can a UTR be generated from a coding region? Does this mean that this is just an alternative translation start site? What does “Default 5’UTR missing, extended downstream from isoform 1” mean? When I think of a gene structure with exons and introns, which splice event would represent this prosaic description? I will not provide more examples here, but by browsing through the supplementary tables I did not find a single useful description. Thus, the authors should provide a gene structure scheme for each gene and mark the events for each gene accordingly. Such schemes would represent exact descriptions of the splicing events. All prosaic descriptions need to be removed.\n=> The authors state in the Methods section: “This was necessary to acquire sequences that resulted from alternative splicing only, as opposed to derivations from other routes.” Which other routes would lead to variants, if not alternative splicing?\n=> The authors state in the Methods section: “A prime example of such a situation occurred with the human database entries, where the newest version contained more verifications than the previous versions.” How did the authors determine, whether the newest version contains “more verifications”? Is there any reference that GenBank obtained more full-length cDNA data in 2016 and that these data were used in the gene predictions?\n=> The authors confirm by experiments the alternative splicing of two Arabidopsis genes, but exactly these variants have already been published by the same group some years ago. Thus the authors do not provide any further and new evidence that any of the predicted variants of the other genes is present and functional. The authors would not even need to do experiments to show functionality. If they only analyse those transcripts for which cDNA/EST data are available, these cDNA/EST data are already the evidence. Further evidence could be obtained if the same transcripts were found for closely related species.\n=> The Introduction is extremely unbalanced. E.g. for this very general statement “Rhomboids are also part of an even larger group of proteins involved in regulated intramembrane proteolysis 2–31.” the authors provide 30! references, but the authors miss to provide a useful description of the protein superfamily in general. As far as I understand, these proteins are present in bacteria, archaea and eukaryotes. It would be essential information how the various subgroups are phylogenetically separated. Without such information, there is no possibility to understand the variants. For example, which of the Arabidopsis homologs are the result of the many plant whole-genome duplications? Do such duplicates have identical alternative splicing? Similar for human and mouse: Which homologs are related to which Drosophila and C.elegans homologs, do the orthologs in human/mouse (derived by the two whole-genome duplications at the origin of the vertebrates) have identical splice variants?\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? 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? No", "responses": [ { "c_id": "3580", "date": "05 Apr 2018", "name": "Kenton Ko", "role": "Author Response", "response": "Thank you for taking the time and effort to review our submission. This is very much appreciated! The feedback provided from the different reports is valuable to us and will guide our revisions for the next version of this contribution. We will be in touch soon with the next version." }, { "c_id": "3667", "date": "31 May 2018", "name": "Kenton Ko", "role": "Author Response", "response": "Dear Dr. Kollmar,Thank you for agreeing to review version 1 of our submission and helping us with your assessment. This is much appreciated! We now have version 2 available for your further assessment. Based on the feedback, Version 2 was revised substantially to address the aspects raised and to enhance clarity for the readers in the areas of writing style and additional details. As per your suggestions, improvements were made to the presentation by removing references that may not be necessary to the readers. Removal of these extra references allowed us to revise the Abstract, Introduction, and Methodology sections to enhance clarity with respect to the study’s focus or purpose. The title was also modified slightly to reflect these revisions.We have revised the Methods section substantially to clarify the approaches used and to provide more needed details concerning the analysis of available database entries and the functionality assays using recombinant proteins. Revisions were also made to the Source Datasets and the Supplementary Tables to enhance their presentation, to provide more descriptive titles and related aspects. We hope that there is now sufficient methodological details for others to follow.The style of writing and presentation in the Methods was revised substantially. We have removed unneeded information and added needed details/information on the tools used, tools that reflect what was being done in the comparative analysis of available entries – namely that we compiled all available entries and assessed their status as splice variants using RNA and protein data. We have added information in the text and in the tables to clarify what the compiled entries represent, e.g., entries or accession numbers listed could represent predicted entries but they are listed and used as reference points after assessment with RNA and protein data.We have added more information (or information deemed missing) concerning the recombinant protein work and revised the information to enhance clarity. For splicing information pertaining to the At1g74130 (L, M, S) and At1g25290 (L, S) protein variants used later in the study, we have added citations and direction to the Supplementary Material for protein information.The multi-year facet of the database work was removed to avoid confusion and to enhance clarity. This removal allowed us to streamline the database analysis protocols and provide more details of the tools used. The Results and Discussion sections were modified similarly to reflect these changes. The removal of the multi-year aspect in combination with the above revisions should help the readers interpret the work more clearly. Concerning the aspect of protein variants with substantial changes being potentially functional, we have provided citations as possible indications that major changes could still give rise to rhomboid proteins with functionality, such as early termination resulting in proteins with one missing transmembrane region.The choice of model organisms used was explained better by revising the style of writing/presentation and by characterizing the work as a comparative analysis or survey of available entries, as opposed to a direct comparison of splice variant numbers between species and its interpretation in this way. We hope that we have interpreted this suggestion correctly. This aspect, we believe, was also raised by Reviewer 3, Dr. Orlov.We did not include references dealing with alternative splicing because the study was not intended to be at this level, but working at the level of surveying and comparing available entries and what these entries may reflect at the protein level. As described above, substantial revisions were focused on clarifying the intent of the work to avoid this impression. This reasoning applies to why gene structures and splicing events were not provided. All of the supplementary tables were revised to enhance the writing style of the descriptions and render them more useful. We hope that the revisions provided clarity on these issues.The latter comments concerning passages in the Methods section were addressed by the revisions outlined above. This would also be the case for the comments pertaining to the Introduction – addressed by the revisions outlined above. Sincerely, Kenton Ko and Josh Powles" } ] }, { "id": "31395", "date": "12 Mar 2018", "name": "Lu-Yuan 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 rhomboids are a very large family of genes found across all kingdoms from bacteria to plants to animals. Based on what we know about the functions of these genes, which unfortunately is not very much at the moment, they often play critical roles in life wherever they are involved. Also interesting is that their functions are highly diversified, as mentioned in this paper. For instance, the human rhomboid family 1 gene, RHBDF1, was found to be expressed at significantly higher levels in breast cancer tissues than in normal breast tissues, with functions as different as promotion of G-protein coupled receptor mediated transactivation of epidermal growth factor receptor (reference 1), or protection of hypoxia-inducible factor 1-alpha from degradation under hypoxic conditions (reference 2); both functions are consistent with facilitating tumor growth, however. RHBDF1 belongs to the group of so-called inactive rhomboids (iRoms) because of an apparent lack of protease activities associated with otherwise enzymatically active rhomboids. This group of rhomboids, having lost their abilities to catalyze proteolysis reactions due to mutations, seem to have destined in evolution to perform other functions, such as those of chaperones, with their abilities to bind to a variety of proteins conserved and utilized in assisting protein folding, transportation, and degradation.\nThe paper by Powles and Ko is interesting as it addresses a fundamental cause of the massive variations of the functions of the rhomboid gene products. The authors carried out an extensive data mining operation to reveal complex differential splicing patterns of the rhomboid genes. Their findings indicate that differential splicing is common and substantial within one gene as well as throughout the gene family. Apart from the multiple transmembrane domains, the rhomboid proteins often possess a large N-terminal domain before the first transmembrane domain and a sizable loop between the first and second ones. The N-terminal and the loop are likely located on opposite side of the endoplasmic reticulum, Golgi, or plasma membrane of the cell, indicating that they have opportunities to interact with different biomolecules. In addition, in the case of RHBDF1 there are a number of amino acid residues within the protein molecule that could be subjected to post-translational modifications such as phosphorylation and glycosylation. It would therefore seem highly likely that frequent and extensive splicing of the gene transcripts exerts considerable impact on the protein structures and thus their functions. Changes in the gene transcripts could also bring about alterations in terms of modulations by micro RNA and other non-translational means. The effort by the authors is very useful to put together a data base of immense and complex differential splicing patterns of nearly the entire rhomboid gene family. The findings should be beneficial to researchers in this field, even though many of the conclusions are speculative, as pointed out by the authors, because of the lack of our knowledge on the structures and functions of most of the individual products of this enormous gene family.\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": "3579", "date": "05 Apr 2018", "name": "Kenton Ko", "role": "Author Response", "response": "Thank you for taking the time and effort to review our submission. This is very much appreciated! The feedback provided from the different reports is valuable to us and will guide our revisions for the next version of this contribution. We will be in touch soon with the next version." }, { "c_id": "3668", "date": "31 May 2018", "name": "Kenton Ko", "role": "Author Response", "response": "Dear Dr. Li,Thank you for agreeing to review version 1 of our submission and for your assessment. This is much appreciated! We now have version 2 available for viewing. Based on the feedback, Version 2 was revised substantially to address the aspects raised and to enhance clarity for the readers in the areas of writing style and additional details. The nature of the work reported was not altered by the revisions.The presentation of the study’s purpose and approach used was revised for clarity. This was achieved first by removing most of the references listed from 2 to 26 so that the references cited were more obviously directed at the study, as opposed to providing general background of the wider field. Accompanying revisions were then made to the Abstract and Introduction.The Methods section was revised substantially to clarify the approaches used and to provide more needed details concerning the comparative analysis of database entries and the functionality assays using recombinant proteins. The title was also modified slightly to align with these revisions. The Results and Discussion sections were modified similarly to reflect the changes. The multi-year facet of the database analysis was deemed unnecessary and removed. Revisions were also made to the Source Datasets and the Supplementary Tables to enhance their presentation. Collectively, these revisions should help the readers interpret the work more clearly with the additional details and streamlining of the protocol for the database work. Sincerely, Kenton Ko and Josh Powles" } ] }, { "id": "31937", "date": "03 Apr 2018", "name": "Yuriy L. Orlov", "expertise": [ "Reviewer Expertise bioinformatics", "genomics" ], "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 manuscript presents original materials on alternative splicing of rhomboid proteins. The authors discuss potential impacts on the rhomboid protein structure. The paper has good illustration and rich Supplementary materials. A lot of work done. Thus, it is technically sound.\n\nBut the presentation has to be improved. A lot of unnecessary literature citations (such as 2-31). Thus, the work does not have clear literature citations.\nChoice of model objects is not clear. Alternative splicing prediction depends on computer tools. If we compare splice variants of the same protein (homolog) from different species, the parameters of prediction should be comparable. Some model organisms, such as human and mouse, have much more experiments and works, and just better gene annotation than for plant genomes. Thus, we can't directly compare number of splice variants. Thus, I consider the statistical analysis and its interpretation only partly appropriate. And not fully reproducible - to answer the question ' Are all the source data underlying the results available to ensure full reproducibility?'\n'Periodic comparison' or 'multi-year analysis' of the information on the proteins family databases should be explained. What is the time for database releases? What kind of trend in functional potential could annotation of these proteins prove? I recommend remove that part of the paper. The study is based on the work by the authors in 2012 on the Arabidopsis thaliana homologs containing alternative splice variants. The current article should show novel materials and conclusion. Comparison by the available information updates on these homologs from model organism databases is a weak idea. If you wait longer, more model species will be functionally annotated, and definitely some new splicing variants found. So, it could be either predictive model, or just description of all data available now on rhomboid proteins.\n'Multi-year analysis' is a redundant term - I recommend remove such part of text, or reformulate it. Overall, the article presents novel ideas on the cross-species comparison of alternative splicing. This science area is growing and we might expect new evidence for alternative splicing from genome sequencing projects. The conclusions drawn by the authors are adequately supported.\nThus, this work is of interest for F1000Research readers. Thus, I recommend 'Approved with Reservations' status.\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?\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? Yes", "responses": [ { "c_id": "3578", "date": "05 Apr 2018", "name": "Kenton Ko", "role": "Author Response", "response": "Thank you for taking the time and effort to review our submission. This is very much appreciated!The feedback provided from the different reports is valuable to us and will guide our revisions for the next version of this contribution. We will be in touch soon with the next version." }, { "c_id": "3669", "date": "31 May 2018", "name": "Kenton Ko", "role": "Author Response", "response": "Dear Dr. Orlov, Thank you for agreeing to review version 1 of our submission and helping us with your assessment. This is much appreciated! We now have version 2 available for your further assessment. Based on the feedback, Version 2 was revised substantially to address the aspects raised and to enhance clarity in the areas of writing style and additional details. As per your suggestions, improvements were made to the presentation by removing references that may not be necessary to the readers. Removal of these extra references allowed us to revise the Abstract, Introduction, and Methodology sections to enhance clarity with respect to the study’s focus or purpose. The title was also modified slightly to reflect these revisions. The choice of model organisms used was explained better by revising the style of writing/presentation and by characterizing the work as a comparative analysis or survey of available entries, as opposed to a direct comparison of splice variant numbers between species and its interpretation in this way. We hope that we have interpreted this suggestion correctly. This aspect, we believe, was also raised by Reviewer 1, Dr. Kollmar. We have revised the Methods section substantially to clarify the approaches used and to provide more needed details concerning the analysis of available database entries and the functionality assays using recombinant proteins. Revisions were also made to the Source Datasets and the Supplementary Tables to enhance their presentation, to provide more descriptive titles and related aspects. We hope that there is now sufficient methodological details for others to follow. The multi-year facet of the database work was removed to avoid confusion and to enhance clarity. This removal allowed us to streamline the database analysis protocols and provide more details of the tools used. The Results and Discussion sections were modified similarly to reflect these changes. The removal of the multi-year aspect in combination with the above revisions should help the readers interpret the work more clearly. Sincerely, Kenton Ko and Josh Powles" } ] } ]	1	https://f1000research.com/articles/7-139
https://f1000research.com/articles/7-685/v1	31 May 18	{ "type": "Case Report", "title": "Case Report: Managing the postoperative exposure of a non-resorbable membrane surgically", "authors": [ "Abdullah S. Almutairi" ], "abstract": "Alveolar ridge deformities can be caused by several factors. Managing alveolar deformities prior to implant placement is essential to increase bone width, height or both. Several techniques and materials are now available to perform ridge augmentation procedures. The postoperative exposure of the membrane is the most frequent postoperative complications of ridge augmentation procedures. The present case describes the horizontal ridge augmentation procedure and the outcome of surgical attempt to manage post-operative membrane exposure, and shows the unpredictability of managing postoperative membrane exposure surgically.", "keywords": [ "Periodontal", "GBR", "membrane", "exposure", "implant", "bone." ], "content": "Introduction\n\nTooth replacement is now easily dealt with using implant placement, which has been a revolution in the past two decades. To place an implant into the alveolar bone, it is imperative to have a sound and stable foundation of bone. Alveolar ridge deformities are very common and may arise due to several causes, including periodontal disease, traumatic extraction, periapical lesions and implant failure1.\n\nAlveolar ridge defects can be classified for diagnosis and treatment purposes. Seibert2 introduced a classification system for ridge deformities. This classified the ridge deformities into three classes according to the horizontal and vertical defect components: class I, horizontal loss of tissue with normal ridge height; class II, vertical loss of tissue with normal ridge; class III, combination horizontal and vertical loss of tissue resulting in the loss of normal height and width2.\n\nNumerous techniques and materials are currently available to manage the resorbed ridge prior to implant placement. Regarding horizontal ridge augmentation, the literature has shown that horizontal bone augmentation is highly predictable, with good resultant implant survival rates3.\n\nThere are several complications related to ridge augmentation, including post-operative membrane exposure, infection, sensory disturbance, additional augmentation procedures needed, and early implant failure4. One of the most frequent postoperative complications of guided regeneration therapy is the membrane exposure5. The present case describes the surgical attempt and its outcome to manage the membrane exposure that had occurred 4 weeks after horizontal ridge augmentation using non-resorbable membrane with particulate allograft bone (mineralized freeze-dried bone allograft (FDBA)).\n\n\nCase report\n\nA 51-year-old male was referred to the Department of Periodontics, College of Dentistry, Qassim University (Buraydah, Saudi Arabia) to extract the non-restorable tooth #45 and to evaluate the site #45 and #46 for the placement of implants.\n\nThe patient had hypercholesterolemia and was taking 20 mg Lipitor (atorvastatin) tablets once daily. Dental history revealed that his lower right first molar was extracted 11 years ago due to caries.\n\nA cone beam CT scan was taken to evaluate the ridge width and height and the location of vital structures (Figure 1 and Figure 2). The radiographic examination revealed deformity of the ridge at site #46 (Siebert class 1). After a discussion with his referring dentist, it was decided to extract tooth #45. A free gingival graft was planned to increase the width of keratinized tissue at site #46 which was followed by ridge augmentation after waiting at least 6 weeks to allow soft tissue healing. The treatment plan was explained to the patient, and written informed consent was acquired.\n\nTooth #45 was extracted and soft tissue healing was completed about 6 weeks later.\n\n6 weeks after the extraction of tooth #45, a free gingival graft was performed to increase the width of keratinized tissue prior to ridge augmentation.\n\nAt 8 weeks after the free gingival graft procedure (Figure 3), ridge augmentation was performed using a titanium-reinforced non-resorbable polytetrafluoroethylene PTFE membrane and FDBA. Local anesthesia with 2% lidocaine and 1:100,000 epinephrine was used to anesthetize the surgical area. A full-thickness mid-crestal incision was made on the edentulous area using a sulcular extension to the distal aspect of tooth #47 and to the distal aspect of tooth #42. A vertical incision was made at the disto-buccal line angle of tooth #42. The flaps were elevated to expose the atrophic ridge (Figure 4). Flap advancement adjacent to the mental foramen area was conducted after a full-thickness mucoperiosteal flap was elevated beyond the mucogingival junction by pushing back the flap using wet gauze until the mental nerve was located (Figure 4). An evident horizontal bone defect was found.\n\nThe mental nerve can be seen.\n\nPeriosteal scoring was performed to release the buccal flap, allowing for coronal advancement of the flap. Decortication was performed in the buccal bone with a round bur (Figure 5).\n\nA titanium-reinforced non-resorbable PTFE membrane (Cytoplast™ Barrier Membranes Ti-250) was stabilized to the buccal plate at the apical end using membrane tacks (Salvin, USA) then FDBA (OraGRAFT®, USA) was placed beneath the membrane and packed gently (Figure 6), then the membrane coronal part was stabilized with two tacks. Flaps were sutured with 4-0 non-resorbable PTFE sutures (Cytoplast™ Sutures, Osteogenics Biomedical) (Figure 7) Postoperative instructions (about diet, pain, bleeding and healing) and medications, including 600 mg ibuprofen three times a day for 5 days, 875 mg amoxicillin twice daily for one week and 0.12% chlorohexidine mouth wash twice a day for 2 weeks, were given to the patient.\n\nAt the 2-week follow up point, the surgical site was healing well, with no sign of infection (Figure 8).\n\nAt the 4-week follow up point, clinical examination revealed that there was post-operative exposure of the membrane. The size of the exposure was approximately 4 × 8 mm (Figure 9 and Figure 10). The sutures were removed and it was decided to manage the exposure surgically by making two small vertical incisions and positioning the tissue coronally to cover the membrane. Non-resorbable sutures 4-0 (Cytoplast™ PTFE) were used (Figure 11). Patient was instructed to use chlorhexidine mouthwash and weekly recall to monitor the surgical site.\n\nAt the 5-week follow up point, 1 week after our surgical attempt to cover the membrane exposure, the size of exposure had increased (Figure 12). Sutures were removed and the outer surface of the membrane was cleaned with cotton swap dipped in chlorohexidine. The patient was instructed to use cotton swab dipped in chlorohexidine to clean the exposed membrane.\n\nAt the 6-week follow-up point, 2 weeks after the membrane covering procedure, intraoral examination revealed pus discharge between the membrane and the tissue (Figure 13). The membrane had to be removed owing to the infection. An incision was made to split them membrane from the tissue then the membrane was removed (Figure 14). After removing the membrane, the underlying tissue had a red, jelly-like appearance, with no bone -graft remnants observed in the surgical site. The flap was sutured using resorbable sutures (Figure 15). The patient was instructed to take 1 g Augmentin twice daily for 1 week and to use 0.12% chlorhexidine mouth wash twice a day for 2 weeks.\n\nNo bone graft remnants were observed in the surgical site.\n\nAt 8 weeks after the horizontal ridge augmentation, the surgical site was healing well with no sign of infection (Figure 16).\n\nAt 5 months after the guided bone regeneration (GBR) procedure, a cone-beam CT scan was performed to evaluate the bone width. The bone width was 9 mm, meaning that the bone width gain after ridge augmentation was 6 mm. two implants were successfully placed at site #46 and #45 (Figure 17 and Figure 19).\n\nThe bone width gain after guided bone regeneration can be noticed.\n\n\nDiscussion\n\nThe purpose of presenting this clinical case was brought out that horizontal ridge augmentation using a combination of titanium-reinforced non-resorbable PTFE membrane and FDBA resulted in the successful implants placement at the sites #46 and #45 despite the membrane exposure that occurred at 4 weeks following horizontal ridge augmentation, the infection that had occurred after a further 2 weeks.\n\nThere are four types of non-resorbable membranes, dense PTFE, expanded PTFE), titanium mesh, and titanium-reinforced polytetrafluoroethylene. In this case, titanium-reinforced polytetrafluoroethylene was used. As with other types of non-resorbable membranes, the most common complication is post-operative exposure6. The rate of membrane exposure following guided bone regeneration is 31%, GBR failure due to membrane exposure have been reported7. Membrane exposure permits a communication between the oral environment and the newly forming tissues, increasing the potential for infection and decreasing the likelihood of regeneration8. In this case, we attempted to manage the exposure surgically by advancing the tissue coronally to cover the exposed membrane, with the aim of preventing the communication between the oral environment and the newly forming tissue. Our second option to manage the exposure was frequent patient follow-up and maintaining good oral hygiene during the healing period.\n\nTo best of our knowledge, no experimental or clinical studies have been conducted to study the proper management of post-operative membrane exposure. In addition to that, there is a disagreement about the impact of membrane exposure on bone regeneration. According to Rita A et al.8 membrane exposure remarkably reduces bone fill, and less bone regeneration around immediate implants sites is observed when exposed membranes are compared to non-exposed membrane sites whereas in a retrospective study where 237 sites treated with guided tissue regeneration were examined, Shanaman9 found that exposure of membrane had no negative impact on bone regeneration if the patient maintained adequate postoperative oral hygiene. The size of exposure increased after our surgical attempt to advance the tissue coronally, however, 1 week after the surgery, no sign of infection was observed which allowed us to keep the membrane in its place. The size of the exposure had increased due to flap sloughing over the membrane owing to inadequate blood supply, which could be explained by the size of the flap and narrow flap base. We did not widen the base of the flap due to the risk of compromising the unexposed membrane. The patient was recalled weekly to check the site. At 2 weeks after the membrane covering surgery, pus discharge between the membrane and the surrounding tissue was noticed. At that time, removal of the membrane was required to avoid the spread of infection to the newly forming tissue. The membrane was removed at 6 weeks after the ridge augmentation procedure. After removing the membrane; the underlying tissue had a red - jelly like appearance with no bone graft remnants observed in the surgical site.\n\nAt 5 months after GBR, the bone width was 9 mm, and eventually, the bone gain was 6 mm, which was assessed with a cone-beam CT scan. Subsequently, two implants were successfully placed at site #46 and #45.\n\nThe present case study shows the unpredictability of managing postoperative membrane exposure surgically. It also, shows that the ridge augmentation was successful after removing the non-resorbable membrane at 6 weeks after the ridge augmentation procedure. Further studies are required regarding the proper management of post-operative membrane exposure.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and 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\nPrato GP, Cairo F, Tinti C, et al.: Prevention of alveolar ridge deformities and reconstruction of lost anatomy: a review of surgical approaches. Int J Periodontics Restorative Dent. 2004; 24(5): 434–45. PubMed Abstract \| Publisher Full Text\n\nSeibert JS: Reconstruction of deformed, partially edentulous ridges, using full thickness onlay grafts. Part I. Technique and wound healing. Compend Contin Educ Dent. 1983; 4(5): 437–53. PubMed Abstract\n\nEsposito M, Grusovin MG, Felice P, et al.: The efficacy of horizontal and vertical bone augmentation procedures for dental implants - a Cochrane systematic review. Eur J Oral Implantol. 2009; 2(3): 167–84. PubMed Abstract\n\nJensen AT, Jensen SS, Worsaae N: Complications related to bone augmentation procedures of localized defects in the alveolar ridge. A retrospective clinicalstudy. Oral Maxillofac Surg. 2016; 20(2): 115–22. PubMed Abstract \| Publisher Full Text\n\nHämmerle CH, Jung RE: Bone augmentation by means of barrier membranes. Periodontol 2000. 2003; 33: 36–53. PubMed Abstract \| Publisher Full Text\n\nSoldatos NK, Stylianou P, Koidou VP, et al.: Limitations and options using resorbable versus nonresorbable membranes for successful guided bone regeneration. Quintessence Int. 2017; 48(2): 131–147. PubMed Abstract \| Publisher Full Text\n\nTomlin EM, Nelson SJ, Rossmann JA: Ridge preservation for implant therapy: a review of the literature. Open Dent J. 2014; 8: 66–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHitti RA, Kerns DG: Guided Bone Regeneration in the Oral Cavity: A Review. Open Pathol J. 2011; 5: 33–45. Publisher Full Text\n\nShanaman RH: A retrospective study of 237 sites treated consecutively with guided tissue regeneration. Int J Periodontics Restorative Dent. 1994; 14(4): 292–301. PubMed Abstract" }	[ { "id": "34962", "date": "16 Jul 2018", "name": "Gerald T. Grant", "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\nAs a case study it does exemplify some of the issues that are seen with bone augmentation and working with membranes. The discussion is appropriate and the observations seem to be accurate.\n\nBoth the photos and the radiographs contribute to the paper as they show the issues as they were addressed.\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": "36237", "date": "09 Aug 2018", "name": "Khalid Almas", "expertise": [ "Reviewer Expertise Periodontology and Implant Dentistry" ], "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 contemporary as far as the scientific writing and the clinical procedures are concerned. The case report is an effort to highlight the complication of ridge augmentation surgery, which is not uncommon. The case has been managed successfully.\nAll the parameters are at par with current routine clinical practice.\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": "36238", "date": "10 Aug 2018", "name": "Zachary Evans", "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 was an interesting report of a common clinical occurrence relating to membrane exposure following oral guided bone regeneration procedures. Some existing evidence suggests that exposure is more common with non-resorbable membranes as seen in this report. This is a very well documented progression showing and discussing the progression of exposure, infection, surgical intervention, and resolution. The report mentions several times the observation of the “red jelly like” substance. This is clearly osteoid that has been formed and not lost due to the infection. Some research is available discussing the speed of formation (1-3 weeks post grafting, but not much is available discussing the formation and stability in the setting of membrane exposure or infection. Therefore, I would recommend this manuscript be considered for indexing with the addition of a brief review or mention of osteoid formation timeline in the discussion.\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/7-685
https://f1000research.com/articles/7-238/v1	28 Feb 18	{ "type": "Research Article", "title": "Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line", "authors": [ "Ekaterina Antonova", "Olga Glazova", "Anna Gaponova", "Aykaz Eremyan", "Svetlana Zvereva", "Natalya Grebenkina", "Natalya Volkova", "Pavel Volchkov", "Olga Glazova", "Anna Gaponova", "Aykaz Eremyan", "Svetlana Zvereva", "Natalya Grebenkina", "Natalya Volkova" ], "abstract": "Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3’-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene (neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.", "keywords": [ "CRISPR/Cas9", "targeting", "homology directed repair", "chicken DF-1 cell line", "endogenous promoter tandem expression" ], "content": "Introduction\n\nGenetically modified chickens have great potential in agriculture, industry, biological research and pharmaceuticals [Farzaneh et al., 2017; Lyall et al., 2011; Nishijima & Iijima, 2013; Schock et al., 2016]. Precise and effective genome editing is one of the most important aspects in creating genetically modified organisms. Traditionally, transgenic chickens were generated using retroviruses [von Werder et al., 2012]. However, retroviral delivery of an inserted sequence is an ineffective method due to random distribution of integration sites, and has adverse effects. Clustered regularly interspaced short palindromic repeat (CRISPR), and CRISPR-associated nuclease (Cas9) - CRISPR/Cas9 are now widely used as an efficient method for genetic modification in a wide variety of organisms [Bassett et al., 2013; Cong et al., 2013; Friedland et al., 2013; Hu et al., 2013; Hwang et al., 2013; Li et al., 2013; Liang et al., 2015; Mali et al., 2013; Nakayama et al., 2013; Niu et al., 2014; Wang et al., 2013; Wagner et al., 2014]. Cas9 cuts double stranded DNA at the site specified by the guide RNA (gRNA). The double strand break can be repaired in an error-prone way by non-homologous end joining (NHEJ), leading to small insertions/deletions, or by homology-directed repair (HDR), when a donor DNA-template is added [Hsu et al., 2014; Merkert & Martin, 2016]. Nuclease-mediated gene insertion is several orders of magnitude more efficient compared with spontaneous recombination of DNA template alone [Lin et al., 2014; Zhang et al., 2017] that makes CRISPR/Cas9 an effective tool for genome editing.\n\nAlthough CRISPR/Cas9-mediated gene editing has been widely used in a lot of organisms, this tool still has been rarely applied in avian species. There are some examples of successfully generated genetically modified chickens with usage of TALEN nuclease (Transcription Activator-Like Effector Nuclease) [Park et al., 2014; Taylor et al., 2017]. Meanwhile CRISPR/Cas9 was used only to knock out genes in poultry [Oishi et al., 2016]. The CRISPR/Cas9 tool is a novel instrument compared with TALEN and some aspects of successful targeting with the Cas9 nuclease still need to be elucidated for avian species. However, the ability not only to knock-out genes, but also to induce a tissue-specific expression of a gene of interest without destroying its endogenous locus would be beneficial.\n\nHere, we show the system to precisely insert a gene under the control of an endogenous promoter and select the cells with the successful integration. The result indicates that this system can be used as a robust tool for chicken genome editing.\n\n\nMethods\n\nWe used human codon-optimized Cas9 (hCas9) as it has been previously demonstrated that the optimized Cas9 works in chicken cells and there was no need to synthesize chicken codon-optimized nuclease [Bai et al., 2016; Véron et al., 2015; Wang et al., 2017; Zuo et al., 2016]. A plasmid CAG-Cas9 (#89995, Addgene; Cambridge MA, USA) was taken for human codon-optimized Cas9 expression. Similarly as for Cas9 it has been previously demonstrated that the human U6 promoter works in chicken cells [Bai Y; Lee et al., 2017; Véron et al., 2015]. Unique 20bp sequences for the selected gRNAs were cloned under human U6 promoter in the plasmid phU6-gRNA (#53188, Addgene). A plasmid pQE30TaqRFP (Evrogen; Moscow, Russia) coding RFP was used for cotransfection as a reporter of the efficiency of DNA delivery to the cells.\n\nThe targeting vector was designed based on the plasmid LSL-Cas9-Rosa26TV (#61408, Addgene). Homology regions of 999bp and 3093bp for left and right arms respectively were amplified from the genomic DNA of chicken cell line DF1 by PCR, and cloned using MauBI and PmeI restriction sites for the left arm, and SgrDI and AscI for the right arm respectively (DF-1 genome is yet to be sequenced, common chicken genomic data is available here). Left and right homology regions in the shuttle flank the P2A-eGFP sequence, where eGFP is enhanced green fluorescent protein. Coding sequence of the left arm was in frame with P2A-eGFP in order to provide the gene transcription under the control of endogenous promoter. Neomycin resistance (neo) gene was cloned after eGFP under a constitutive cytomegalovirus (CMV) promoter for positive selection of cells with the desired insertion. The total length of the inserted sequence between two homology arms in the shuttle was 3259bp. Diphteria Toxin Fragment A (DTA) coding sequence was inserted in the shuttle after the right arm under a PGK promoter for negative selection (Figure 1A). The left homology arm was amplified by PCR with the following primers: 5’-TTGTTGACCTGACCTGCCGTCTGGAG-3’ and 5’-CTCCTTGGATGCCATGTGGACCATCAAG-3’; the right homology arm was amplified with primers 5’-CCCTTTGTTGGAGCCCCTGCTCTTC-3’ and 5’-GAGCCCTGTATCTTCCTTGCACAGACC-3’. The primer were designed in Primer-BLAST and synthesized by Evrogen.\n\n(A) A schematic illustration of the chicken GAPDH locus, HDR-cassette, edited GAPDH locus. (B) A schematic diagram of the target sites in the chicken GAPDH 3’UTR flanked with homology arms.\n\nLength of the right homology arm is longer in order to increase HDR due to high repeat content in 3’URT region. Chicken right and left homology arms are separated by a stretch of 42bp in the beginning of 3’UTR that is targeted by the gRNAs (Figure 1B). Thus, CRISPR/Cas9 cutting of successfully edited genomic DNA is prevented due to the targeted sequence not being present in the vector for HDR.\n\nWe searched for gRNAs to target the GAPDH locus in the 3’UTR within 50bp near the stop codon of the gene. We sequenced exon 10 of GAPDH and the beginning of the 3’UTR. 100bp region of the chicken GAPDH around the stop-codon was analyzed on the ChopChop server for gRNA design. We selected three gRNAs in the locus (Table 1, Figure 1B).\n\ngRNAs target sequences and PAMs are shown. gRNA - guide RNA; PAM - Protospacer adjacent motif.\n\nThese gRNAs had a few predicted off-target sites (Table S1). None of the off-target sites were present in a known coding sequence of the Gallus gallus genome.\n\nThe chicken DF-1 cell line (ATCC, CRL-12203) was cultivated in DMEM medium (Gibco, ThermoFisher) containing 10% fetal bovine serum (FBS, Gibco) and 100u/ml penicillin/streptomycin mix according to ATCC recommendation. Cells were maintained at 37°C with 5% CO2. For experiments, DF-1 cells were plated into 6-well or 24-well-plates.\n\nCells were co-transfected with 4µg DNA plasmid mix for 6-well plates or 1µg for 24-well plates using TurboFect transfection reagent (ThermoFisher; Waltham MA, USA). The plasmid mix contained the Cas9 plasmid, the gRNA plasmid, the RFP plasmid and the linear HDR shuttle. We used the equimolar ratio of all components: pCas9:pgRNA:pRFP:HDR shuttle. After 72 hours post transfection, cells were analyzed by fluorescence microscopy. Five – seven technical repeats were made for every experiment, which were used for genome analysis, G418 selection and for flow cytometry analysis.\n\nTitration of geneticin (G418, Gibco) on the DF-1 cell line had been performed before selection and the optimal concentration 500ng/µl was chosen. Cells were cultured with geneticin for 10 days with daily medium change. Geneticin-resistant cells were analyzed with PCR to confirm the correct insert. Fluorescence microscopy and FACS were used to visualize and define the percentage of eGFP-positive cells respectively.\n\nFor evaluation of an appropriate concentration of geneticin, CellTiter-Blue® Cell Viability Assay (Promega, Fitchburg WI, USA) was used. The principle of the assay is based on conversion of resazurin to resorufin by metabolically active cells that results in the generation of a fluorescent shift from 605 nm to 573 nm. Thus, the produced fluorescence is proportional to the number of viable cells. 48-well assay plate containing cultured cells with media was set up. Three technical repeats were performed for each concentration at each time point. G418 (Gibco) in concentrations of 100ng/µl; 200ng/µl; 500ng/µl; 1000ng/µl; 2000ng/µl and 5000ng/µl was added. The recommended volume of CellTiter Blue Reagent was added to a series of wells at 48, 96, 140, 192 and 240 hours following geneticin treatment. After addition of the reagent, cells were incubated for 2 hours. Fluorescence was measured (CLARIOstar - BMG Labtech; Offenburg, Germany) at 560/590nm. 570–600nm absorbance versus concentration of G418 was plotted.\n\nFor detection of Cas9-mediated cuts, a T7E1 assay was performed [Kim et al., 2009]. The assay is based on the ability of the T7 Endonuclease to recognize and cleave non-perfectly matched DNA. DF-1 cells were harvested and genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit (Promega). PCR products were amplified using the primers T7-for: 5’-GACCATTTCGTCAAGCTTGTTTCC-3’ and T7-rev: 5’-GATCAGTTTCTATCAGCCTCTCCCAC-3’. The primer were designed in Primer-BLAST and synthesized by Evrogen. The amplified product, 635bp in length, was purified from agarose gel. 200ng of the product was reannealed to form heteroduplex DNA at the following temperature conditions: 95°C – 5min; 95°C-85°C with ramping 2°C/sec; 85°C-25°C with ramping 0,1°C/sec; 25°C – 2min; 4°C. - After the reannealing, T7 nuclease EI (New England Biolabs; Ipswich MA, USA) was added (10 units). Heteroduplex DNA was incubated with the enzyme at 37°C during 30 min. The resulting product was analyzed by electrophoresis.\n\nMutation frequencies were calculated as described by Guschin et al. (2010) based on the band intensities. Band intensities were measured with ImageJ software (version 2). Mutation frequency (%) = 100 · [1 – (1 – F)1/2], where F represents the cleavage coefficient, which is the proportion of the total relative density of the cleavage bands to all of the relative densities of the cleavage bands and uncut bands [Guschin et al., 2010].\n\nTo confirm HDR cassette integration several pairs of primers were used (Figure S1D). The primers were designed based on schematic sequence of the edited locus in Figure S1D, checked in BLASTn for specificity and synthesised by Evrogen.\n\nPrimers forward: 5’-GACCATTTCGTCAAGCTTGTTTCC-3’ and reverse: 5’-GATCAGTTTCTATCAGCCTCTCCCAC-3’ amplify the area surrounding the site of insertion (Figure S1D, primers pointed as 1’ and 1’’). PCR product in the case of insertion had a length of 3851bp. Amplified product from the endogenous locus without an integration had a length of 635bp.\n\nAmplification with primers exon 2-forward: 5’ - AATGGGCACGCCATCACTATCTTC - 3’ and P2A-reverse: 5’ - TGGCCCGGGATTCTCTTCGA - 3’ results in product only in the case of successful HDR-mediated cassette integration (Figure S1D, primers pointed as 2). Primers forward 5’-GACCATTTCGTCAAGCTTGTTTCC-3’ and reverse 5’-tggcccgggattctcttcgac-3’ (Figure S4D, primers pointed as 3) were used for PCR analysis from the genome of cells enriched by drug selection after successful modification. The product is only amplified in case of an unmodified genome. This happens due to the reverse primer aligning to the 50bp area of 3’UTR that was targeted by gRNAs, but it does not align to the HDR vector itself.\n\nFACS analysis (Accuri™ C6 Flow Cytometer, BD Biosciences; San Jose CA, USA) was used to estimate the proportion of eGFP- positive cells.\n\nPotential CRISPR/Cas9 off-target sites for selected guides are presented in Table S1. The off-targets were predicted by ChopChop tool. All off-target sites were located in introns or in intergenic loci.\n\nPrimers for in vitro gRNA synthesis:\n\nIn order to make dsDNA template for RNA transcription the two following oligonucleotides were annealed.\n\n-CRISPR R: (common primer for all targets, it contains the sequence for RNA scaffold synthesis) - the oligonucleotide was common for synthesis of all DNA templates coding a gRNA:\n\n5’–AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC–3’-CRISPR F: (the oligonucleotide contains the T7 promoter and the specific target sequence). Both oligonucleotides have a 20nt complementary sequence that allows them to anneal.\n\nCRISPR F GAPDH gRNA1\n\n5’–GAAATTAATACGACTCACTATA GGGGCATCCAAGGAGTGAGCC GTTTTAGAGCTAGAAATAGC–3’\n\nCRISPR F GAPDH gRNA2\n\n5’–GAAATTAATACGACTCACTATA GGGTGTGCCTGGCTCACTCCT GTTTTAGAGCTAGAAATAGC–3’\n\nCRISPR F GAPDH gRNA3\n\n5’–GAAATTAATACGACTCACTATA GGGCTTCCCTAGGCAGCAGGG GTTTTAGAGCTAGAAATAGC–3’\n\nThe oligonucleotide synthesis was ordered from Evrogen.\n\nFull-length dsDNA template was made via PCR overlap of corresponding oligonucleotides\n\nHiScribe T7 High Yield RNA Synthesis Kit (E2040S, New England Biolabs) was used for in vitro RNA synthesis of gRNAs, as described in the manufacturer’s protocol. RNA was purified using the MEGAclear™ Transcription Clean-Up Kit (AM1908, ThermoFisher) following the protocol from the manufacturer.\n\nIn vitro digestion was made by Cas9 Streptococcus pyogenes (S. pyogenes; New England Biolabs) according to the protocol from the manufacturer. PCR product for analysis was amplified with following primers: forward 5’-GACCATTTCGTCAAGCTTGTTTCC-3’and reverse 5’-GATCAGTTTCTATCAGCCTCTCCCAC-3’.\n\nDroplet digital PCR (ddPCR).\n\nProbes description\n\nWe designed three kinds of probes. Probe 1 - a reference probe (VIC) located away from the editing site to count all genomic copies (Figure S2I). GAPDH was used as a reference, having a single copy per genome [Weiskirchen R., et al., 1993].\n\nProbe 2 - a (FAM) probe, located in the inserted sequence (Figure S2I). Probe 3 - a (VIC) probe located in DTA gene of the cassette that does not insert into the locus or kill the cells in case of insertion (Figure S2I). The nucleotide sequence of the probes and primers are shown in the Table 2.\n\nSamples description.\n\nThe following samples were used:\n\n-DNA from DF1 cells transfected with linearised cassette only (negative control). The sample was taken in amount 20ng.\n\n-DNA from geneticin enriched DF1 cells, isolated 1 month after HDR. The sample was taken in amount - 1ng and 20ng.\n\n-Water was used as a no template control (NTC) to rule out cross-contamination.\n\nWe set up two repeats for each amount of DNA.\n\nCassette probe was used to determine the copy number of transgene in DF1 cells. GAPDH probe was used as the reference.\n\nddPCR reaction preparation.\n\nThe following reagents were mixed in 96 plate to make reaction:\n\nddPCR Supermix for Probes (#186-3026, Bio-Rad);\n\n10 U of EcoRI (#R3101S, New England BioLabs);\n\nGenomic DNA (DNA dilutions 1ng and 20ng were selected based on preliminary experiments);\n\nThe total volume of the reaction was 20µl.\n\nDroplets were generated with 20μl of the premixed reaction and a QX200 Droplet Generator according to the manufacturer’s instructions (Bio-Rad) and transferred to a 96-well PCR plate for standard PCR on a CFX96 Touch™ Real-Time PCR Detector system (Bio-Rad).\n\nThe following cycling programs were used:\n\n1) 95°C for 10 min;\n\n2) 95°C for 30 s;\n\n3) 59–63 °C (in depends on pair of primers and probe) for 1 min; repeat steps 2 and 3 for 40 times;\n\nOptimal annealing temperature was determined empirically for each pair of primers and probe with a temperature gradient. After PCR amplification, each droplet provides an independent fluorescent positive or negative signal indicating the target DNA was present or not. The droplets were analysed with a QX200 Droplet Reader (Bio-Rad) with the selected option “absolute quantification”. Positive and negative droplets are counted for each samples, and the software calculates the concentration of target DNA as copies per microliter.\n\nQuantification of ddPCR data.\n\nQuantaSoft (version 1.7.4.0917) was used for quantification (Bio-Rad).\n\nAn appropriate threshold between the positive and negative droplets was applied manually based on the NTC wells. ddPCR software reads the positive and negative droplets in each sample and plots the fluorescence droplet by droplet. The fraction of positive droplets determines the concentration of the target in the sample. Software calculates the concentration of target DNA as copies per microliter. Then the copy number of an unknown target is calculated relative to a known reference. In our case we estimated the copy number of inserted sequence relative to GAPDH gene. The confidence intervals for each well are calculated by QuantaSoft based on Poisson distribution.\n\nThe formula used for Poisson modeling is:\n\nCopies per droplet = –ln(1 – p)\n\nwhere p = fraction of positive droplets.\n\nThe primers and probes were designed in Primer-BLAST. Primers were synthesised by Evrogen. Probes were ordered from Syntol (Moscow, Russia)\n\n\nResults\n\nIn the current research we have studied CRISPR/Cas9-mediated homology directed repair in an endogen locus for expression of an integrated gene under the control of the endogenous promoter.\n\nThe GAPDH locus was chosen as a commonly expressed constitutive gene. In order to make the expression of our inserted gene be controlled by the promoter, we decided to insert the gene just after the coding sequence of GAPDH (Figure 1A). To target the 3’UTR of chicken GAPDH we selected and designed three gRNAs. Before starting experiments on a chicken cell line, all selected gRNAs were tested in vitro using recombinant Cas9 S. pyogenes. Cas9 in complex with one of the three gRNAs made cuts and produced lengths of cleaved products corresponded to the expected lengths (Figure S3).\n\nIn order to test the effectiveness of targeting endogenous 3’UTR of GAPDH with the selected gRNAs in chicken cells, Cas9, gRNA plasmids, combined with the RFP plasmid, were co-transfected in DF-1 cells. Cas9 expression vector without a gRNA was used as the negative control. RFP expression was estimated at 72h after transfection (Figure S4). Figure S2 is represented by seven technical repeats. The average level of transfection efficiency was more than 45%.\n\nGenomic DNA was extracted from the cells, and T7 endonuclease I (T7EI) assay demonstrated that only gRNA2 in complex with Cas9 had activity in DF1 cells with the targeting rate around 1.8% (Figure 2).\n\nIn order to obtain cells with the insertion we co-transfected DF-1 cells with plasmids encoding Cas9, gRNA, RFP and cassette for homology directed repair. As a negative control we added Cas9, RFP and cassette without any gRNAs. At 72h after transfection we observed 0.5% GFP-positive cells of transfected cells in the experimental group (Figure 3). Figure 3 is represented by five technical repeats. In the vector for HDR, eGFP does not have its own promoter and can be expressed only in the case of the correct insertion in frame with the GAPDH gene. The genomic DNA was extracted for PCR analysis. The analysis confirmed the presence of the expected integration (Figure S1A and Figure S6B).\n\n(A) Transfection with gRNA2, Cas9, RFP; (B) Transfection with gRNA2, Cas9, RFP, cassette for HDR; (C) Transfection with Cas9 without any gRNA, RFP, cassette for HDR. Scale bar = 200 µm.\n\nApplying drug selection in combination with CRISPR/Cas9 allowed us to select and grow colonies carrying the modification. Based on the survival curve (Figure S5) we added 500 ng/µl of geneticin (G418) at 72h after transfection. The appropriate concentration of the drug was selected after titration in the DF-1 cell line (Figure S5). Single eGFP-positive cells had developed in colonies after 10 days of incubation on G418. RFP fluorescence had vanished due to its transient expression (Figure 4). Figure 4 is representative of five technical repeats.\n\n(A) 72h after transfection; (B) 15 days after selection; (C) 1 month after selection. Scale bar = 200 µm. Figure is representative of five technical repeats.\n\nOne month after the drug selection the enriched cells were analyzed by PCR and FACS. PCR analysis confirmed the absence of cells without modification (Figure S1C). FACS analysis showed 90% of eGFP positive cells in the cell population (Figure S6).The nucleotide composition around the insertion was analyzed by DNA sequencing, additionally confirming the correct integration (Figure S7). Also we made a ddPCR analysis to measure the copy number of the integrated sequence per genome. DNA samples from enriched edited cells was analysed in comparison with DNA sample from the cassette only transfected cells (at 72 hour after transfection). 1-D plot with FAM positive droplets (Figure S2.II.A), indicating insertion, and VIC positive (Figure S2.II.B) droplets, indicating the reference gene GAPDH, plotted on the graph of fluorescence intensity versus droplet number is shown for each sample (please see the method description). The positive droplets determines the concentration of the target in the sample which are calculated into the concentration of target DNA as copies/µl. The number of copies of the insertion was normalised by the number of copies of GAPDH as it is known that birds have one copy of the gene [Weiskirchen et al., 1993]. In result we got about 0,8 - 1 copy of the insertion per genome (Figure S2.III) that additional confirm the FACS data. The copy-number of DTA gene was also estimated to differentiate the cassette or improperly integrated sequence from the correct insertion. In the enriched edited cells DTA gene was not observed (data is not shown in the Figure S2.II). Thus, all used methodological approaches confirmed the correct-ness and effectiveness of the transgene integration.\n\n\nDiscussion\n\nCRISPR/Cas9 is easy to use, specific, efficient, and multiplex [Cong et al., 2013; Mali et al., 2013]. Here, we set up a system for efficient CRISPR/Cas9-mediated homologous recombination to successfully target chicken DF-1 cells. The approach can be used to obtain cell populations with a gene of interest under the control of a tissue-specific promoter. In this study, we targeted the 3’UTR area of chicken GAPDH. Successful attempts to target chicken genome with CRISPR/Cas9 have been reported. Mammalian codon-optimized Cas9 has been used to target PAX7 gene in chicken somatic cells [Véron et al., 2015], PPAR-g, ATP5E, OVA genes [Bai et al., 2016], myostatin gene [Wang et al., 2017] in DF-1 cells, C1EIS gene [Zuo et al., 2017] and Stra8 gene [Zhang et al., 2017] in male germ cells. Mammalian codon-optimized Cas9 and chicken U6 promoter for gRNA were also used to target C2EIP gene in DF-1 and chicken embryonic cells [Zhang et al., 2017; Zuo et al., 2016]. The researches demonstrated activity of mammalian adapted CRISPR/Cas9 in avian cells and the ability to knock out a gene. The CRISPR/Cas9 targeting efficiency in our experiments, according to T7E1 assay analysis, was around 1.8% that is similarly with the results from the previous article [Bai et al., 2016]. The low effectivity in our case can be explained by several reasons: we were restricted by 50–100 bp sequence of genomic DNA for selection of gRNAs; the area of targeting is the beginning of 3’UTR, which usually has a lower GC content and has a lot of repeated elements; also the transfection efficiency of the DF1 cells with multiple plasmids was less than 50%.\n\nTraditionally homology directed recombination with long homology arms was used to insert a desired sequence at a desired place. For example, homology directed recombination at chicken JH segment was performed without CRISPR/Cas9 using ~8–9 kb of total homology arms. The inserted sequence had a size ~2000bp. The frequency of the insertion was low, about one targeted clone per 107 transfected cells, and after drug selection it resulted in 28% of correctly targeted events [Schusser et al., 2013]. Thus, the approach has low effectiveness and accuracy.\n\nIt is known that applying Cas9 with a vector for homologous recombination enables the usage of 2kb of total homology instead of 7–8kb significantly increasing the effectiveness of an insertion. Several approaches to make homology directed repair in chicken cell line using CRISPR/Cas9 has been recently published. A stable genetic element in the chicken genome of the DF-1 cell, endogenous avian virus (EAV-HP), was targeted and the inserted sequence was 1200bp length [Wang et al., 2017]. The EAV-HP is considered as a safe harbor, and can be used to generate constitutive expression of a gene of interest, although the element is contained in the chicken genome in multiple copies. The targeted effectiveness reached 49%. In the other article the Cas9 system was used for modifying the variable domain of the immunoglobulin heavy chain (IgH) in chicken PGCs in vitro [Dimitrov et al., 2016]. The authors targeted a site approximately 300bp upstream of the translation initiation site of IgH using homology regions of 1133bp and 1011bp with the inserted sequence around 1500bp and had 33% of effectiveness after drug selection. Therefore, the researches demonstrated higher effectiveness of CRISPR/Cas9-mediated homology directed repair for constitutive expression of the inserted gene in chicken cells compared with traditional methods.\n\nIn our study we inserted into the GAPDH 3’UTR region a sequence up to 3000bp, that encoded the eGFP gene expressed under the control of an endogenous promoter and Neomycin resistant gene controlled by the CMV promoter. The sequence of 3’UTR and the following downstream sequence is not well characterized in chicken models [International Chicken Genome Sequencing Consortium, 2004], that potentially could reduce the opportunity to design long homology regions required for a traditional gene targeting approach, so the CRISPR/Cas9 system is very helpful in the case. The length of homology regions has to correlate with the size of the insertion and we used homology regions 1000 and 3000bp. In practice amplification and cloning of the homology arms od such length was not labour-intensive and the same strategy of targeting a tissue-specific gene can be easily applied to other loci of interest. In our experiments HDR effectiveness at 72 hours after transfection was around 0.5% of RFP-positive cells in the case of targeting with gRNA2. Using drug selection, we achieved up to 89% targeted integration.\n\nExpression from endogenous promoters could be favorable for other applications, for instance, synthesis of pharmaceutical proteins in the egg white under the ovalbumin promoter, or expression of a gene that provides a defense against a pathogen in tissues that are located in contact with the infection. For now, genome modification in chickens has been established using germline stem cells, such as primordial germ cells (PGCs) [Leighton et al., 2008; Song et al., 2014; van de Lavoir et al., 2006; van de Lavoir et al., 2006]. We are planning to use our approach to insert a gene of interest, in place of eGFP, under the control of the tissue-specific promoter of PGC, and enrich cells after homology directed repair. Cultured PGCs can be transfected and injected into recipient-embryos, where they will produce the germline.\n\nIn conclusion, we demonstrated that the CRISPR/Cas9 system along with cassette for HDR can successfully target the 3’UTR of endogenous genes to integrate a gene under endogenous control.\n\n\nData availability\n\nDataset 1: Raw images of all gel images (Figure 2 and Supplementary Figure S1 and Figure S3) 10.5256/f1000research.13457.d192397 [Antonova et al., 2018a]\n\nDataset 2: Plate reads for all time points performed for the cell viability assay (Figure S4) 10.5256/f1000research.13457.d192398 [Antonova et al., 2018b]\n\nDataset 3: FACs output files underlying Figure S5 10.5256/f1000research.13457.d192408 [Antonova et al., 2018c]\n\nData underlying Figure S8 is available from Dataverse: http://dx.doi.org/10.7910/DVN/YSNKKC [Antonova, 2018d]\n\nAvailable under a CC0 - \"Public Domain Dedication\"", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe work has completed under the financial support of the Russian Science Foundation within project No. 16-16-04094.\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\nTable S1. Predicted off-targets for each gRNA.\n\nClick here to access the data.\n\nFigure S1. Confirmation of correct insertion with PCR. (A) The result of PCR analysis with primers 1’ and 1”; (B) The result of PCR analysis with primers 2; (C) The result of PCR analysis with primers 3; Scheme of GAPDH locus before and after HDR-mediated insertion with primers. 1. Genomic DNA from cells, transfected gRNA, Cas9; 2. Genomic DNA from cells, transfected gRNA, Cas9, HDR cassette; 3. Genomic DNA from cells, transfected Cas9 without gRNA, cassette; 4. H2O; 5. Cassette; 6. Genomic DNA from eGFP-positive 1 month since geneticin selection.\n\nClick here to access the data.\n\nFigure S2. I. The scheme of genomic DNA before and after the cassette insertion with marked location of primers and probes for ddPCR. Probe 1 – VIC (green), Probe 2 - FAM(blue), Probe 3 - VIC(green). II. Representation of ddPCR results in 1-D plot for the following samples: Ex 20 ng DNA, Ex 1 ng DNA – DNA from DF1 cells isolated 1 month after HDR experiment following G418 selection; Control 20ng DNA – negative control, DNA from DF1 cells transfected with linearized cassette only; NTC- no template control. (A) Amount of FAM positive droplets indicating the presence of the insertion. (B) Amount of VIC positive droplets. VIC probes target GAPDH used as a reference to determine the copy number of insertion per genome. Positive droplets are those above the pink threshold line. III. The copy number of the insertion (transgene) per genome. Experiment - DNA from DF1 cells cultivated during one month after HDR in presence of G418; Control – negative control, DNA from DF1 cells transfected with linearized cassette only.\n\nClick here to access the data.\n\nFigure S3. In vitro Cas9 digestion, using gRNA1, 2 and 3. Cas9 with non-targeting scramble gRNA and Cas9 without any gRNA were used as negative controls.\n\nClick here to access the data.\n\nFigure S4. Evaluation of plasmid delivery efficiency in GAPDH targeting experiment. (A) Cas9, gRNA1, RFP transfection; (B) Cas9, gRNA2, RFP transfection; (C) Cas9, gRNA3, RFP transfection.\n\nClick here to access the data.\n\nFigure S5. Results of cell viability assays for G418 using the CellTiter-Blue Reagent.\n\nClick here to access the data.\n\nFigure S6. Geneticin selection of eGFP-positive cells by flow cytometry analysis.\n\nClick here to access the data.\n\nFigure S7. Sequencing of integrated HDR vector in the modified locus of GAPDH. (A) The coverage of sequenced area. (B) Sequencing confirms in frame eGFP insertion.\n\nClick here to access the data.\n\n\nReferences\n\nAntonova E, Glazova O, Gaponova A, et al.: Dataset 1 in: Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line. F1000Research. 2018a. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "31286", "date": "12 Mar 2018", "name": "Zhiying Zhang", "expertise": [ "Reviewer Expertise Genome editing 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\nEkaterina Antonova, et al. reported their successful integration of \" P2A-eGFP-CMV-NeoR \" cassette in chicken DF-1cells, achieving eGFP expression under control of the endogenous GAPDH promoter. As the authors claimed, their approach may be used to insert genes of interest under control of tissue-specific promoters for further producing genetically modified birds. The experiments were well designed and the paper was well written.\n\nHowever, there are still some concerns:\n\n1. Page 3, in the Introduction section, the authors claimed that \"Meanwhile CRISPR/Cas9 was used only to knock out genes in poultry\". Actually, HDR-based gene editing and integration have also been reported previously as the authors cited (Wang et al.,2017, Dimitrov et al., 2016; page 10) in the Discussion section.\n\n2. Although the guide RNA (gRNA) is also named single guide RNA (sgRNA), it would be better for the authors to use gRNA or sgRNA consistently through the whole paper.\n\n3. Page 7, the authors claimed \"FACS analysis showed 90% of eGFP positive cells in the cell population(Figure S6)\", but the data as Figure S6 shown was \"EGFP+, 88.1\". Nevertheless, page 10, in the Discussion section, the authors again claimed \" Using drug selection, we achieved up to 89% targeted integration\". In my opinion, they mean the same data. Please check it.\n\n4. The in vitro assay demonstrated all 3 gRNAs functioned (Figure S3), but the T7E1 assay suggested only gRNA2 functioned in cells (Figure2). Thus, the in vitro assay is of no sense and is no longer needed. As a suggestion, the authors may use surrogate reporters 1,2 for future validation of gRNA activity.\n\n5. Besides, the off-target effect of gRNA2 was not assessed.\n\n6. As the authors discussed in the Discussion section (page 9), \" transfection efficiency of the DF1cells with multiple plasmids was less than 50%\". Regarding that the Cas9 gene, the U6-gRNA expression cassette and the RFP marker gene can be easily cloned together within one single plasmid, why did the authors use 3 separate plasmids?\n\n7. One more concern. The bi-allelic integration in mammalian cells is usually very low. However, here both the PCR and ddPCR analysis implied that all copies (alleles) of GAPDH gene were integrated at the 3'-end with the intent eGFP cassette, after the enrichment.\n\nAlthough the authors described that \" as it is known that birds have one copy of the gene (GAPDH, page7)\", I think it is a pair of alleles but not a single GAPDH gene. Thus, I am wondering that how the mono-allelic integration became bi-allelic after the selection? By HR and the untargeted allelic will gain the integration of eGFP from the targeted 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? 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": "3681", "date": "30 May 2018", "name": "Ekaterina Antonova", "role": "Author Response", "response": "1. The sentence: \"Meanwhile CRISPR/Cas9 was used only to knock out genes in poultry» is about researches that resulted in generation of transgenic chicken (F1 from gremlin chimera) with application of CRISPR/Cas9 technology. Still there are no data about a genetically modified bird with a gene integration performed with the CRISPR/Cas9. Wang et al.,2017 made research with CRISPR/Cas9-mediated integration in cell culture and Dimitrov et al., 2016 obtained gremline chimeras. 2. We named all guide RNAs as gRNA through the paper as you recommended. 3. Yes, it is the same data. We rounded the number consistently in the page 7 and the discussion section. 4. We will try surrogate reporters in future researches as you advised. 5. Yes, however we made a bioinformatic analysis and found that none of the off-target sites were present in a known coding sequence of chicken genome. Thus experimental off-target estimation is not so actual for the guides. 6. Three separate plasmids were used to optimise several conditions that can influence on gene targeting efficiency. We tested different ratios of Cas9 and gRNA. Also we tested amplicons and plasmids for gRNAs delivery. The best conditions were selected for HDR and were presented in the article. RFP was used for cotransfection. We agreed with you that usage one plasmid with all components could increase the number of transfected molecules of instruments per cell and thereby could increase targeting effectiveness. Alternatively mini-circles can be used for the same purpose. 7. We mentioned about one copy of the gene, because genomes of other animals have different number of GAPDH pseudogenes [Yuen-Jong Liu et al., 2009]. However one copy of GAPDH implies two alleles. PCR analysis does not allow to answer the question bi-allelic or mono-allelic integration happened, the amplicon of the exact size can be amplified in any case of the correct insertion. Based on ddPCR data we calculated the ratio of copies of transgene per one allele of GAPDH gene in total. In the figure S2.III there is a misprint in the title. It should be “Transgene copies per GAPDH allele” instead “Transgene copies per genome”. The title was modified in the version 2. EGFP-positive cells and Geneticin resistant cells could have monoallelic or biallelic integration. The ratio of transgene per genome is 1,6±0,4 copies based on ddPCR analysis." } ] }, { "id": "32664", "date": "02 May 2018", "name": "Lei Qi", "expertise": [ "Reviewer Expertise genetic engineering", "synthetic biology" ], "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 work is a confirmation that the CRISPR/Cas9 system and a template DNA design can be technically used in a chicken cell line for inserting a reporter gene into a genomic locus. The authors demonstrated that insertion of an EGFP gene cassette into the 3’UTR of the endogenous GAPDH gene with an efficiency of 0.5%, and 89% with selection with antibiotic. The authors provided a detailed description of the protocol, which is helpful for other researchers to follow. However, the work remains a proof-of-principle demonstration that the CRISPR+DNA donor can work in a chicken cell line, the efficiency is likely not high enough for applications. The authors didn’t spend efforts to improve the efficiency, nor investigate what might be the causes of the low efficiency. The protocol used in the chicken cell line is largely taken from the one that is used in mammalian cells, including the human codon optimized Cas9, and mammalian promoters for expressing Cas9 and gRNA. To make a convincing case that their method is “robust”, they should test inserting more genes (not only a reporter gene) into more genomic loci (not only GAPDH). Because of this, it remains a question if the described method is robust enough for inserting any gene of interest into any genomic locus of interest. The suggested next-step experiments include optimizing the method to increase the efficiency, testing more genomic loci, testing gene insertion with different lengths, and testing in the chicken primodial germ cells that will be useful for the applications. Other suggestions for improvement:\nWhile the authors demonstrate inserting a promoterless gene into the genomic under the control of an endogenous promoter, they also claimed potential tissue-specific gene expression. However, they didn’t prove that their method can result in a cell line that exhibits tissue-specific gene expression.\n\nThe efficiency of gene cutting in vitro is lower than expected, only 1.8%. What causes this low efficiency? In general, the authors should provide a detailed discussion of the generally low efficiency observed both in vitro and in vivo, which will be the primary targets for improvement.\n\nThe authors should mention one additional advantage of their use of the endogenous promoter to express inserted genes, which is less likely be epigenetically silenced. From their data, they observed a relatively long-term (1 month) expression from the endogenous GAPDH promoter. This can be an advantage for long-term stable expression of pharmaceutical proteins compared to using an exogenous promoter that can be epigenetically silenced over time.\n\nThere is not off-target assay to characterize the real off-target sites from the computational ones. Also, what is NHEJ rate in the correctly EGFP inserted cells? Are there alleles also show indel editing via NHEJ? It would be useful to understand the NHEJ vs. HDR landscape in a chicken cell line.\n\nThe authors should try additional experiments to improve the efficiency. For example, they can try to reduce the number of plasmids used for transfection, test methods such as RNP delivery or using Cas9 mRNA, use a chicken codon-optimized Cas9 and chicken promoters to express Cas9 and gRNA, and re-design the 5’ and 3’ homologous arms. Via these experiments, it may become a more useful resource to understand what might lead to better HDR efficiency in a chicken cell line.\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": "3687", "date": "30 May 2018", "name": "Ekaterina Antonova", "role": "Author Response", "response": "1. Yes, we tell that potentially the method can be used for integration in a tissue-specific locus. The next step will be to apply the approach for integration under a germ cell-specific promoter in PGC cells. 2. The low efficiency can be explained by several reasons: we were restricted by 50–100 bp sequence of genomic DNA for selection of gRNAs; the area of targeting is the beginning of 3’UTR, which usually has a lower GC content and has a lot of repeated elements; also the transfection efficiency of the DF1 cells with multiple plasmids was less than 50%. The 1,8% effectiveness was pointed for T7E assay but not for in vitro assay. The following simple steps could be done for improvement of the locus targeting in the cell line: (for increasing CRISPR/Cas9 targeting:) -Targeting the last exon and mutation of PAM in HDR cassette instead of targeting the beginning of 3'UTR in the case when there are no appropriate predicted gRNAs. -Increasing of transfection effectiveness (usage of all-in one vector or minicircles, usage of chicken U6 promoter, usage of RNA/RNP delivery for the instruments, etc). (for HDR improvement:) -Usage of longer or smaller homology arms. The optimal length of arms could vary in depends on targeting locus. -Blocking NHEJ pathway in order to increase HDR events. 3. The advantage was mentioned in the Version 2, the Discussion section: “Usage of an endogenous promoter to express an inserted gene also could prevent its epigenetic silencing. This is very important for long-term stable expression of pharmaceutical proteins and other applications.” 4. Yes, however from computational analysis we found that none of the off-target sites were present in a known coding sequence of chicken genome. For instance, gRNA2 targets intragenic locus. Thus experimental off-target estimation is not so actual from point of view of probable harm. Regarding NHEJ and NHEJ vs.HDR: We did not estimate NHEJ rate in the correctly EGFP inserted cells, we used ddPCR only for HDR calculation. We can not analyse the NHEJ vs. HDR landscape like in the publication [1], due to different design of the experiment. In our study long homology arms and long integrated sequences were used that did not allow us to estimate HDR and NHEJ simultaneously. Different integrations might occur at two genomic alleles in a single cell. Some EGFP+ cells could have the correct knock-in in at least one allele. Another allele can be unchanged or can contain NHEJ. 5. There are a lot of parameters for changing in order to improve gene targeting efficiency. Some of them has been already optimized for chicken cells in literature. For instance, U6 promoter yields 4-fold higher gRNA expression than the human U6 in DF1 cell line [2]. We have tested gRNAs delivery in plasmids or amplicons in the cell line, Cas9 with different nuclear localisation signals (SV40 and nucleoplasmin) and several ratios of the instruments [data not shown]. The best conditions were taken to be presented in the article. The aim of the study was not to optimise Cas9 mediated targeting. We wanted to demonstrate that the approach with negative and positive selection works in chicken cells allowing to obtain modified cell line even when targeting effectiveness is low that is expected in the case of PGC cells. We suppose that the results can be improved. For example, variation of different sizes for the 5’ and 3’ homologous arms could increase the quantity of desired cells before enrichment etc. We agree with you that our method can not be called as a robust. It is a possible way for a gene insertion under control of a desired endogene promoter in chicken cells. In further researches the methods should be applied in PGC cells. [1]. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing Yuichiro Miyaoka, Jennifer R. Berman, Samantha B. Cooper, Steven J. Mayerl, Amanda H. Chan, Bin Zhang, George A. Karlin-Neumann & Bruce R. Conklin. [2]. Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo. ShashankGandhiMichael L.PiacentinoFelipe M.VieceliMarianne E.Bronner" } ] } ]	1	https://f1000research.com/articles/7-238
https://f1000research.com/articles/7-675/v1	29 May 18	{ "type": "Opinion Article", "title": "Life experience and the asymmetry of the human auditory system: clinical and auditory science laboratory implications", "authors": [ "Dale Hewitt" ], "abstract": "It is now almost 60 years since Doreen Kimura first described the asymmetries of the human auditory system (HAS). It is 30 years since Kenneth Hugdahl, and almost 15 years since David Poeppel, did the same. Our knowledge of these asymmetries is now considerable and detailed. Here we review some of the literature concerning what is known about the asymmetry of the HAS, and use it to construct a simple model of how the central and peripheral components work together. The model details some of the asymmetry, the life-experience-dependant maturation of this asymmetry throughout the first two decades, possible reasons for the population variance in speech-in-noise perception, the central role of the corpus callosum, the involvement of the efferent auditory pathways, and the corticofugal control of the peripheral auditory system. Although our knowledge of HAS asymmetry remains incomplete, this should not prevent the introduction of required changes to the current practices of audiologists and auditory science researchers, practices which fail to recognize the existence of any asymmetry in the HAS. The model described here suggests a clear need for: “asymmetry sensitive” hearing test tools that provide normative data for the lifespan, closer childhood and aging-adult hearing monitoring, and the development of a range of auditory training modules. Notably, the model suggests that without such tools our HAS asymmetry knowledge will remain incomplete. The status quo is a preoccupation with understanding hearing through knowledge of the peripheral hearing system, no answers for those with a “normal audiogram” but with an obvious difficulty hearing speech-in-noise, limited success cochlear implant rehabilitation, and missed opportunities to successfully intervene with childhood hearing/speech/language development problems.", "keywords": [ "Audiological rehabilitation", "auditory plasticity", "auditory training", "childhood development", "dichotic listening test", "hidden hearing loss", "right ear advantage", "speech-in-noise recognition" ], "content": "Introduction\n\nHere we review some of the literature concerning the asymmetry of the human auditory system and use it to construct a model of how the central and peripheral components work together. The model uses the Doreen Kimura “structural model” of dichotic listening as its foundation [see Kimura, 1961; Kimura, 2011], and is augmented by the findings of the auditory scientists who have continued the work she began nearly 60 years ago.\n\nThe progress of audiological rehabilitation and auditory science research remains compromised by the continuing preoccupation with understanding hearing through knowledge of the peripheral hearing system [Hewitt, 2018; Musiek et al., 2017]. Only when the depth of our knowledge of the central auditory system matches that of the peripheral auditory system, and audiological practice is informed by the integration of this knowledge, can we expect to properly rehabilitate those with hearing problems.\n\n\nThe Doreen Kimura “structural model” of dichotic listening\n\nThere are ascending auditory pathways that connect both ears to both the left and right Auditory Cortices (ACs) (Figure 1). However, as Doreen Kimura discovered [Kimura, 1961; Kimura, 1967; Kimura, 2011], the primary connections are the crossed pathways. Normal hearing involves the right ear feeding sounds to the left AC and the left ear feeding sounds to the right AC.\n\nThe right ear feeds sounds to the left auditory cortex (AC) and the left ear feeds sounds to the right AC using the crossed ascending pathways.\n\n\nThe Bergen dichotic listening test with consonant-vowel syllables\n\nIn her research. Doreen Kimura made extensive use of various dichotic listening (DL) tests. One of the most popular dichotic listening tests is the Bergen dichotic listening test with consonant-vowel (CV) syllables (DL-CV) [Hugdahl & Andersson, 1986; Hugdahl & Asbjørnsen, 2013]. Each running of the Bergen DL-CV test involves 30 presentations using headphones. Each presentation involves the simultaneous playing of two randomly chosen CV syllables—one to the right ear and one to the left ear. The test uses only six CV syllables—all of them stop consonants combined with the /a/ vowel: /ba/, /da/, /ga/, /pa/, /ta/, /ka/. There are three DL-CV test runs, of 30 presentations each. The first run is the non-forced condition. This is then followed by the forced-right condition and then the forced-left condition. An option is to reverse the order of the forced conditions. Figure 2 includes the instructions given to test participants for the three different conditions.\n\nAC, auditory cortex.\n\nFor all three DL-CV test conditions, a correct report (%) is calculated for both the right ear and the left ear. Figure 3 shows the right and left ear scores that are typically found in DL-CV studies with normal hearing participants. It also includes the typical non-forced condition scores found for studies of participants who have had a callostomy—a surgical procedure involving some level of cutting of the corpus callosum so as to limit the spread of epileptic activity between the two hemispheres of the brain [see Musiek & Weihing, 2011].\n\nHigher non-forced condition (NF) scores for the right ear than for the left ear. This difference increases with the forced-right condition (FR) scores and decreases with the forced-left condition (FL) scores. For those who have had a callostomy, the right ear has almost no competition from the left ear. (Data is contrived.)\n\nThe results for studies involving individuals with normal hearing typically show higher non-forced condition scores for the right ear than for the left ear. This difference increases with the forced-right condition scores and decreases with the forced-left condition scores. This many repeated set of findings has become known as the “right-ear advantage (REA)”. However, we should be careful to be more specific and refer to these findings as showing a “REA for CV syllables” (REA-CV). Doreen Kimura conducted many different dichotic listening studies [Kimura, 2011] and as well finding a “REA for digits”, also found a “left-ear advantage (LEA) for melodies” and a “LEA for non-verbal sounds”. Following on from this work Ley & Bryden [1982] found a “LEA for the emotional tone of speech”. The REA-CV finding is therefore only one of many dichotic listening study findings which demonstrate the asymmetry of the two hemispheres of the human brain and auditory system. The REA-CV is a result of the right ear being “directly” connected by the crossed pathway to the left AC, and the left AC being more specialised at phoneme processing. As the left ear is only “indirectly” connected to the left auditory cortex (via the contralateral pathway, the right AC and the corpus callosum) then it is at a disadvantage with a dichotic test involving CV syllables [Kimura, 2011]. By contrast, the left ear has the advantage with a dichotic test involving emotionally intoned sentences. This is because of the “direct” connection of the left ear to the right AC by the crossed pathway, and the right AC being more specialised at prosody processing. We can refer to this as the LEA-emotion or LEA-prosody.\n\n\nThe corticofugal modulation of the asymmetrical auditory system\n\nTaken on their own, the non-forced condition scores for the REA-CV and the LEA-prosody reflect bottom-up processing advantages. However, the forced condition results (Figure 3) show that top-down driven attention to one of the ears is able to modulate the scores. In the case of attention to the right ear this equates to increasing the REA-CV, and in the case of attention to the left ear it equates to decreasing the REA-CV, and in some individuals even reversing the advantage altogether. Westerhausen & Hugdahl [2008] describe this as two components working together; one “in-built” and bottom-up that is stimulus driven, the other attention driven and top-down, that enables modulation of the “in-built” asymmetry. The Bergen DL-CV Test has been used by many different studies over many years. Kenneth Hugdahl and his team at Bergen University, Norway developed the test, and have used it as a research tool for over 30 years [Hugdahl et al., 2009; Westerhausen et al., 2015]. The Bergen Dichotic Listening Database [Westerhausen et al., 2015] and the resulting normative data provided with their test manual [Hugdahl & Asbjørnsen, 2013] enables an understanding of the effects of age upon the REA-CV. There has been little discussion of these age effects in the literature but they provide valuable insight into the lengthy maturity window of the human auditory system through childhood and adolescence, and also its gradual decline, beginning as early as the sixth decade (Figure 4).\n\nNormative data obtained from the ‘Bergen Dichotic Listening Test with CV-Syllables Manual’ [Hugdahl & Asbjørnsen, 2013].\n\nTaking the effects listed in Figure 4 in turn:\n\n1. The AC asymmetry seems to be “in-built” and lasts a lifetime\n\n2. If we think of the ability to use top-down attention to modulate the in-built asymmetry of the auditory system as a skill, then it seems that this corticofugal skill does not begin to develop until the end of the first decade, but it then continues to develop into adulthood\n\n3. There is evidence (see below) which shows that the interhemispheric pathways of the corpus callosum play significant roles in the deterioration of the left ear scores (and in the development of the above corticofugal skill)\n\nA Finnish version of the Bergen DL-CV test was developed by researchers at the University of Turku, Finland and was used in their studies concerning childhood development [see Takio et al., 2009]. They too found that the top-down (corticofugal) skill to modulate the asymmetry of the auditory system does not begin to develop until the second decade. In a related study, this time in collaboration with Kenneth Hugdahl, they looked at the enhanced auditory processing skills of the congenitally and early blind [Hugdahl et al., 2004]. They found, as they predicted, that the blind subjects were significantly better than the seeing (control) subjects at using their top-down corticofugal skill when instructed to attend to the left ear stimulus. Using the REA-CV test, Hugdahl & Andersson [1987] found that, in a group of children aged from 8 to 9 years, there was a clear connection between increasing reading skills and increasing top-down corticofugal skills. In a related study, Andersson & Hugdahl [1987] found slower maturation of the corticofugal skill in a group of eight year old boys compared to a group of similarly aged girls.\n\nLooking at the dichotic listening test results of adult individuals, rather than adult population averages, reveals that some test participants are able to entirely reverse the REA-CV when undertaking the forced left condition. Others are only able to increase their left ear score by small amounts, with the right ear score remaining considerably higher than the left ear score. The Bergen normative data [Hugdahl & Asbjørnsen, 2013] shows that there is a large between-individual variability. This leads to the proposition that, with some individuals, the skill to be able use top-down attention to modulate the asymmetry of the auditory system fails to properly develop.\n\nAn understanding as to why there is such a large between-individual variability is suggested by the dichotic listening test studies that looked at the differences between the DL-CV test scores of musicians and non-musicians. Milovanov et al. [2007] found that both choir-members and non-musical adults had similar REAs with the non-forced condition NF. The non-musical adults still had a clear right ear advantage with the forced left condition. The choir-members however, had a clear LEA with the forced left condition. The musicians had developed the skill to moderate the asymmetrical auditory system, while the non-musicians had not.\n\n\nThe nurturing of hearing skills\n\nSeveral different auditory science research teams have conducted many different music related auditory studies in recent years. Most of these have concluded that with children (and young adults) there is a strong relationship between the extent of music practice and the enhancement of neural responses to speech, and as a consequence, better hearing speech-in-noise recognition\n\nSee, for example, the review paper of Strait & Kraus [2014]. These studies additionally found that the auditory training effects of music continued until the end of adolescence [see Krizman et al., 2015]. The REA-CV literature reveals the same finding, and in Figure 4 we can see that the difference between the forced and non-forced scores (both for the left ear and the right ear) is minimal at first, increases through adolescence and into early adulthood, and then becomes stable during adulthood. Tonal pitch processing is an important component of musical perception [Zatorre et al., 2002] and can be measured using the scalp-recorded frequency following response (FFR). Coffey et al. [2017a] found that the strength and fidelity of the FFR correlates well with speech-in-noise recognition scores. Also see Du et al. [2011]. They also found that the effect was stronger with the right AC than with the left AC. With young adults, Coffey et al. [2016] found a strong, right-asymmetric contribution to the FFR from the human auditory cortex, and that the magnitude of the response was related to musicianship. It is well accepted that musical training enhances abilities related to such things as pitch, rhythm and melody. However, there does remain some disagreement in the literature about the benefits of music experience skills with regards to improved recognition of speech-in-noise. Madsen et al. [2017], for example, found that although musicians showed better fundamental frequency discrimination, this did not translate into better speech-in-noise recognition in their study. However, in their review of the results of similar studies they did find examples of a clear speech-in-noise recognition benefit of musicianship. These particular studies differed from the others (and their own) in that speech-in-noise recognition was measured using a test that spatially separated target speech from noise.\n\nThe vast majority of the speech-in-noise studies in the literature describe their use of speech audiometry tests that use headphones. Such tests do not represent realistic listening situations, and do not test speech-in-noise skills that relate to the asymmetry of the auditory system [Coffey et al., 2017a; Hewitt, 2018]. Nevertheless, a recent review concerning the speech-in-noise recognition advantages of musical training was still able to conclude that, with 18 out of 20 studies showing an effect, musicians are better at hearing speech-in-noise [see Coffey et al., 2017b].\n\n\nThe MOC system and top-down control of the peripheral hearing system\n\nSeveral research studies concerning the connections between music and speech processing have focussed upon the top-down driven control of peripheral hearing by the medial olivocochlear (MOC) system, and the enhanced MOC systems of those who have undertaken musical training. Bidelman et al. [2017] found that musically trained individuals show enhanced ipsilateral and contralateral cochlear gain control, and that there is a correlation between MOC strength and length of training. Musical training strengthens dynamic MOC activity, and speech-in-noise recognition is described as a likely benefit of the enhanced corticofugal control of the peripheral hearing system shown by musicians [Perrot & Collet, 2014]. Using DL-CV testing, Markevych et al. [2011] found that those with greater MOC strength were better at using top-down corticofugal skill to overcome their REA-CV when instructed to attend to the Left Ear stimulus.\n\n\nChildhood development and the in-built asymmetry of the ACs\n\nRecent studies made possible by non-invasive neurophysiological techniques have enabled closer inspection of the parallel processing by the two ACs during childhood. The evidence suggests that even though asymmetry is in-built, the asymmetry is relatively immature at birth, matures through childhood and adolescence, and is not mature until early adulthood.\n\nAri-Even Roth et al. [2016] found, as have many other otoacoustic emission based studies, that the transient-evoked otoacoustic emission results of a newborn hearing screening programme demonstrated that auditory system asymmetry is already apparent shortly after birth. Using event-related potential (ERP) techniques Musacchia et al. [2017] found that in the first year of life, early targeted acoustic experience can accelerate the maturation of both temporal and spectral processing. Using magnetoencephalography, Tang et al. [2016] showed that, compared to adults, the sound envelope following response was limited in capacity in children 3 to 5 years of age. Thompson et al. [2016] studied children 3–5 years of age to examine the evidence for temporal asymmetry early in life. They found a leftward asymmetry for higher-frequency oscillations and that this was more pronounced for those who scored higher on a speech-in-noise test. However, they did not find any rightward asymmetry for the lower-frequency oscillations. They proposed that this was a possible consequence of the known slower development of both white and grey matter structures in the right AC, and that this reflects the greater immaturity of the right AC at this young age. Clunies-Ross et al. [2018] studied temporal processing in children aged 7 and 9 years and, as expected, found evidence of left hemisphere specialisation (and a right hemisphere bias for spectral processing). They suggest that at this age, as the specialisation was not as prominent as with adults, the full extent of the asymmetry has yet to be reached, and that the immaturity of the right AC is greater. Using magnetoencephalography, Nora et al. [2017] studied children 6 to 8 years old and found that, compared to adults, the left hemisphere has yet to become dominant with regards (native language) phonological processing. They propose that at this age the learning of new words involves the use of the prosodic processing of the right hemisphere. Yathiraj & Vanaja [2015] showed that, in children between 6 and 10 years, different auditory processes matured at different rates. By measuring cortical responses, a study by Yamazaki et al. [2018] showed that, between the ages of 5 and 15 years, there is increasing specialisation of the two ACs. Using ERP, Mahajan & McArthur, [2013] studied left and right hemisphere auditory processing in adolescents aged between 10 and 18 years and found that they seem to mature at different rates, with the latter taking longer and continuing well into adolescence. In their longitudinal study using auditory brainstem response, Krizman et al. [2015] identified change taking place in auditory brainstem function between ages 14 and 17. They concluded that compared to the first decade different kinds of maturation take place in the second, and that the beginning of adolescence marks a transitional point.\n\nWe have described how human hearing develops throughout the first two decades of life and involves a slow but ever-increasing specialisation of the two auditory cortices. The related binaural skill of sound source localisation has also been found to gradually develop throughout childhood, adolescence and into early adulthood [see Freigang et al., 2015; Glyde et al., 2013; Grothe et al., 2010]. Spierer et al. [2009] studied brain-damaged patients and showed that auditory spatial representations for both left and right hemispaces are a function of the mature right hemisphere. At et al. [2011] reported the same finding in a study that used single pulse transcranial magnetic stimulation (to temporarily alter normal brain function). The Freigang et al. [2015] study includes a description of the age effects on sound localisation; with minimum audible angle (MAA) scores for 8–12 year olds not as mature as the adult-like MAA scores of 13–18 year olds. Glyde et al. [2013] describe how the many studies that have used the listening in spatialized noise-sentences test (LiSN-S) to measure the ability to process the sound localisation cues (measured by the LiSN-S “high-cue” score) have shown spatial processing continuing to mature until early adulthood.\n\nThe two decade maturation of AC specialisation and sound source localisation is prohibited by single-sided deafness (SSD). An understanding of SSD outcomes therefore enables consideration of the possible outcomes of a binaural auditory system that fails to fully mature. Zhang et al. [2018] found that unilateral hearing impairment lasting longer than 24 months drove cortical functional changes including enhanced interhemispheric AC connectivity and altered connectivity with visual and somatosensory networks. In a review, Eggermont [2017] concluded that, irrespective of the age of induction of a single-sided hearing loss, several hearing system adaptations take place, including modified interhemispheric connectivity and the near disappearance of the contralateral dominance of the ascending pathways. These adaptations take place over a few months and this short timescale can even mean that these adaptations will affect some individuals (both children and adults) who suffer from persistent otitis media with effusion (OME). Using REA-CV testing, Asbjørnsen et al. [2000] found that children who had undergone a myringotomy for persistent OME (at an earlier age) were less able to modulate the REA-CV advantage. Jafari et al. [2016] reported similar findings. Gordon et al. [2013] found that after 18 months of unilateral hearing with a cochlear implant that there was a loss of the contralateral dominance of the ascending auditory pathways and that this maladaptation still remained even after 3–4 years of subsequent use of bilateral cochlear implants (see also Polonenko et al. [2017]). Gordon et al. [2015] provide a summary of the hearing problems caused by SSD and some of the consequences. These include difficulty in hearing speech in noisy situations, impaired speech and language development, reduced verbal IQ, and the frequent outcomes of behavioural problems and requirement for individualized educational assistance.\n\n\nThe role of the corpus callosum\n\nIn their review of the role of the corpus callosum in dichotic processing Musiek & Weihing [2011] concluded that dichotic listening scores relate to the physical development of the corpus callosum. Dichotic listening performance improves until adolescence matching the increasing size of the corpus callosum. In their review paper Westerhausen & Hugdahl [2008] describe how the corpus callosum plays a pivotal role in the central auditory system. Sammler et al. [2010] show that the posterior pathways of the corpus callosum are involved in the integration of the syntax processing of the left AC and the prosody processing of the right AC. In a review, Homae, [2014] concluded that during early years, the processing of auditory stimuli changed from independent ACs to mutually inhibitive ACs and modified corpus callosum interhemispheric connectivity. As Musiek & Weihing [2011] describe, some of the interhemispheric connections (between the two ACs) resulting from the maturation process are excitatory, others are inhibitory, and the maturation involves an increased rate of transfer of information (as a consequence of fibre myelination). In a review paper Bamiou et al. [2007] describe how the corpus callosum size increases until the third decade of life and then decreases again after the fourth decade. They also suggest that brain lateralization depends upon fast inter-hemispheric transfer, and that the corpus callosum has an important role in the processing of binaural cues and spatial hearing. In their recent review of the white matter asymmetries of the nervous system Ocklenburg et al. [2016] concluded that, although further investigation is still needed to understand the detail of the pathways and the inhibition and excitation involved, the hemispheric functional asymmetries depend upon the CC and its maturation path. The importance of the corpus callosum to auditory processing means that, as it “normally ages”, it also plays a pivotal role in what has become known as central presbycusis. The review by Musiek & Weihing [2011] reports that the worsening of left ear scores in dichotic listening tests begins in the middle of the sixth decade, as the corpus callosum begins to reduce in size (see also Figure 4).\n\n\nDiscussion\n\nWe have learnt about how the peripheral auditory system and central auditory system work together by looking at dichotic listening literature and the auditory science literature. In the dichotic listening literature review we re-examined and re-interpreted the effects of age data, and then went on to look at the different DL-CV test scores of musicians and those with no sight. With the auditory science literature review we focussed upon the relationship between musical training and auditory processing skills, the enhanced corticofugal control of the individual cochlea by musicians, the slowly maturing specialisation of the two auditory cortices, and the pivotal role of the corpus callosum in the increasing AC asymmetry during the first two decades of life. Additionally, the SSD literature was examined to consider the possible outcomes of a binaural auditory system that fails to fully mature. The findings of this set of literature reviews are highly consistent and complementary, and have led to a set of conclusions about the human auditory system.\n\nThe human auditory system has evolved to be capable of performing the real-time processing of complex acoustical stimuli such as speech and music. Some individuals can even manage this task in the presence of competing background noises. With these individuals, it seems that they have specially trained their auditory system through more frequent binaural intensive and challenging listening experiences. This extensive training seems to result in increased asymmetry, increased neural enhancement, and greater corticofugal control of the individual cochlea. With greater maturity, the sense of hearing has been divided into two, with the left AC responsible for temporal processing and the right AC responsible for spectral processing [also see Sininger & Bhatara, 2012; Zatorre et al., 2002], and spatial representation. The processing of stop consonants for example requires millisecond differences to be detected and is a function of the left AC. The changing melody of musical notes occurs over seconds and its processing is a function of the right AC. When listening to speech-in-noise, these individuals are able to use parallel processing, with the heavy myelination of corpus callosum fibres enabling fast interhemispheric communication between the two ACs. To enable speech recognition in noise, right AC neuron populations (measured using the scalp-recorded frequency following response) are phase-locked onto the low-to-middle-frequency periodical acoustical features of the sound source [Du et al., 2011], sound source locations are more precise, and corticofugal control of OHCs is able to modulate cochlea sensitivity and to restore lost dynamic range [Perrot & Collet, 2014]. By contrast, the adult outcome for those who are limited to small numbers of binaural intensive listening experiences during the first two decades, is an untrained and immature auditory system. They are unable to use parallel processing, the phase locking to an individual sound source is imprecise, they have little corticofugal control of the OHCs, and they struggle to understand speech in the presence of background noise. In sum, while some asymmetry exists at birth, it is life experience that shapes the detail of the auditory processing capability and the functional asymmetry of an individual's auditory system.\n\nThe auditory system maturation window seems to be open from birth until the beginning of adulthood with the maturation taking place during the first decade relating more to the temporal processing specialisation of the left AC. The maturation taking place in the second decade appears to relate more to the right AC and its spectral processing and sound location specialisations. A significant outcome of the maturation during the second decade is the establishment, or not, of corticofugal control of the peripheral auditory system, via the descending efferent pathways. The review paper by Terreros & Delano [2015], as well as discussing the MOC control of the OHCs, describes the recent findings concerning the efferent control of the inner hair cells (IHCs) of the cochleae via the lateral olivocochlear (LOC) neurons. Their working model proposes that there are three afferent-efferent feedback loops that enable the dynamic corticofugal modulation of the IHCs and OHCs. They describe the LOC/MOC control of the IHCs/OHCs as a top-down frequency filter that facilitates, for example, speech recognition in noisy environments.\n\n1. The mature auditory system is to some extent asymmetrical and with some individuals the two ACs are capable of parallel processing\n\n2. With regards the processing of speech, the asymmetry equates to a left AC (right ear) responsible for phoneme processing and a right AC (left ear) responsible for prosody processing. Temporal (phoneme) processing mostly matures during the first decade of life, whereas spectral (prosody) processing takes longer to mature. The mature right AC is responsible for the neural representation of both the left and right hemispaces.\n\n3. The more extensive and challenging the auditory listening experience leading up to adulthood, with regards the need to simultaneously process both temporal and spectral features, the greater the asymmetry, the greater the neural enhancement, and the more refined the auditory processing skills\n\n4. Musical training is known to improve sensory representation, sequencing skills, working memory, auditory attention, stream segregation, and top-down expectations; resulting in “musicians” having superior speech perception in noise ability, compared with “non-musicians” [Chandrasekaran & Kraus, 2010]\n\n5. Very different childhood/adolescent listening experiences mean that auditory processing capability and the extent of asymmetry varies considerably—between children, between adolescents, between young adults, and between adults. These differences are “completely hidden from” and not measurable using standard audiometry tools (Hewitt, 2018; Musiek et al., 2017)\n\n6. With some individuals top-down attention is able to modulate auditory system asymmetry. This corticofugal “skill” is well-developed in some adults but hardly at all in others. It appears connected with the maturation of the prosody processing of the right AC and its development occurs (or not) during the second decade of life.\n\n7. The corticofugal “skill” involves all of the auditory pathways. This includes the ascending afferent pathways, the two ACs, the interhemispheric pathways of the corpus callosum, the descending efferent pathways (including the olivocochlear efferent fibres), and ultimately the dynamic control of the hair cells of the two cochlea (see Figure 5).\n\nThe right ear feeds sounds to the left auditory cortex (AC) and the left ear feeds sounds to the right AC, using the crossed ascending pathways. When listening to speech, the left AC is responsible for phoneme processing and the right AC is responsible for prosody and spatial processing. The corpus callosum interhemispheric pathways connect the two ACs together. Top-down attention can modulate the asymmetry and involves the descending (efferent) pathways and the dynamic control of the hair cells of the two cochlea.\n\nThe “structural model” of Doreen Kimura [Kimura, 1961; Kimura, 2011] was a precursor to the “asymmetric sampling in time” (AST) model [Poeppel, 2003]. The AST model equally aims to provide a framework for understanding the asymmetry of the function of the two ACs. It describes them as anatomically similar but functionally specialised, with the left AC adapted to extracting information from shorter (20–40 ms) time windows, and the right AC adapted to extracting information from longer (150–250 ms) time windows. In attempting to aid the understanding of the asymmetry of the human auditory system this article shares the objectives of the Doreen Kimura “structural model”, the David Poeppel AST model, and the series of Hugdahl et al. and Westerhausen et al. dichotic listening studies (Westerhausen & Hugdahl [2008] provide references to most of these). In a related review Tervaniemi & Hugdahl [2003] describe the lateralisation differences between the encoding of speech and the encoding of music. This article, however, has an objective over and above an updated review of our knowledge concerning the asymmetrical auditory system—that of translating the knowledge into the availability of improved rehabilitation in the audiology clinic.\n\nThe original articles of Kimura [1961]; Kimura [1967] and Poeppel [2003] have been cited thousands of times in the literature and yet, now nearly 60 years since the first of these articles, audiological rehabilitation continues to fail to recognize the existence of any asymmetry in the human auditory system [Hewitt, 2018; Musiek et al., 2017]. One important outcome concerns speech audiometry. With a lack of consideration of the binaural nature of hearing and the asymmetry of the central auditory system, most speech audiometry tools are unable to determine speech-in-noise recognition capabilities reliably [Hewitt, 2018]. Their lack of usefulness has meant little use in the audiology clinic and their use restricted to auditory science research laboratories. Most of these tests use headphones and “one ear” testing (see, for example, Hewitt [2008]). Such tests involve the perception of sound inside the head, do not represent realistic listening situations, do not involve spatial cues, do not test the speech-in-noise skills that relate to the asymmetry of the auditory system, and do not test prosody (e.g. emotion recognition) [Coffey et al., 2017a; Hewitt, 2018]. As a consequence there are limitations to the validity of the results and conclusions of the auditory science research studies that have used such “one ear” speech testing. This is the case with the majority of the hearing research published in the academic literature, with the validity of the results of those studies concerned with speech recognition in noise being more in question (see also Phatak et al. [2018]).\n\nThe model of the asymmetrical human auditory system (and those of the past) leads us to the conclusion that changes to the current practices of both audiologists and auditory science researchers are needed. The suggestions included in this and subsequent sections of this article are intended to stimulate discussion about the required nature of these changes.\n\nGreater knowledge of how human hearing works improves our abilities to assess hearing, to diagnose hearing problems and to manage those with hearing difficulties. The model suggests, for example, that there is a need for:\n\n1. Wider-scope hearing test tools (for the audiology clinic and auditory science laboratory) that involve the “exercising” of both cochlea, both auditory afferent pathways, both auditory cortices, the corpus callosum, and both auditory efferent pathways.\n\n2. Hearing test tools that provide normative data for the lifespan (i.e. separate normative data for children, adolescents, young adults, adults, and old adults).\n\na. Not unlike height and weight, auditory system development during childhood requires close monitoring, with the appropriate interventions used when developmental problems are identified.\n\nb. Auditory system aging (presbycusis) requires close monitoring, with the appropriate interventions used sooner rather than later (see also Glick & Sharma [2017]).\n\n3. The development of auditory training modules (ATMs), for the lifespan (i.e. different ATMs for children, adolescents, young adults, adults, and old adults).\n\nThe simple model described in Figure 5 generates as many questions as it answers; the following are only some of these questions.\n\nThe model supports the use of ATMs for those with hearing problems based on the premise that “real life” hearing training for subsets of the population, such as musicians or the blind, bestows auditory skills such as speech recognition in noise. Assuming that practical (and perhaps personalisable) ATMs can be developed then there is a prospect that they will be able to play a significant role in:\n\nThe rehabilitation of cochlear implant recipients [see Anderson & Kraus, 2013]\n\nThe management of those suspected to have Auditory Processing Disorder [see Moore et al., 2018; Weihing et al., 2015]\n\nChildhood Speech and Language development [see Clunies-Ross et al., 2018]\n\nAnd perhaps they hold out the promise of:\n\nEffective speech perception in noise for the majority of adults\n\nProlonging hearing skills into old-age [see Strait & Kraus, 2014]\n\nDelaying the aging of the auditory system and cognitive decline [see Hewitt, 2017; Humes & Young, 2016; Livingston et al., 2017; Lin et al., 2013; Loughrey et al., 2018]\n\nCan a form of dichotic listening test be developed that will reliably and efficiently predict speech recognition in noise ability? There is a case for using the existing REA-CV test as a screening tool. Also see Asbjørnsen et al. [2000] and Jafari et al. [2016].\n\nUnfortunately, unlike with the DL-CV studies, the literature does not provide significant amounts of data from the use of dichotic listening-prosody testing. Would such data provide further insight into the slower development of the right AC?\n\nIt is well accepted that standard pure tone audiometry (PTA) is unsuitable for measuring speech in noise recognition capability [Musiek et al., 2018]. More recently it has been shown to be insensitive to cochlear synaptopathy (CS). CS can result from excessive noise and from aging. Standard PTA is unable to detect CS until it becomes extreme, and as a consequence the condition has become known as “hidden hearing loss” [Kujawa & Liberman, 2009; Liberman et al., 2016]. Ipsilateral (“one ear”) speech-in-noise testing was used by Liberman et al. [2016] to compare a group of people with normal sensitivities up to 16 kHz with a group of people with normal sensitivities up to 8 kHz but threshold elevation for the 10–16 kHz range. The latter showed worse speech-in-noise discrimination scores. The study concluded that to increase the possibility of CS detection, both high-frequency audiometry and speech recognition testing should be used in the audiology clinic with greater regularity. The model of the asymmetrical auditory system leads us to proposed extensions to the recommendations made by Liberman et al. [2016]. While the more commonly used speech audiometry tools are designed for ipsilateral presentation of speech and noise to a single ear using headphones, a small number of sound field speech audiometry tools are currently available that use loudspeakers for spatially separated speech and noise presentation. Although normative data is perhaps limited, such sound field speech audiometry tools enable some degree of measurement of actual binaural speech-in-noise recognition capability. Therefore:\n\nTo increase the sensitivity to hearing loss not detectable using standard PTA, high-frequency audiometry and sound field speech audiometry testing should be used in the audiology clinic with greater regularity.\n\nTo increase the sensitivity to speech-in-noise recognition capability, sound field speech audiometry testing should replace headphone based (“one ear”) speech audiometry in the auditory science laboratory.\n\nSound field speech audiometry additionally supports “before and after” hearing aid evaluation [Hewitt, 2018].\n\nTo improve speech-in-noise recognition outcomes when fitting hearing aids, less reliance should be placed upon the results of PTA scores, with more reliance placed upon “before and after” sound field speech audiometry results\n\nGordon et al. [2015] reached a similar conclusion: that standard paediatric audiology clinic test procedures require modification, as they fail to be sensitive to the increased risk of educational difficulties that result from single-sided hearing. See also Musiek et al. [2018].\n\n\nConclusion\n\nThis article extends a listening model first published nearly 60 years ago. The extended model describes a human auditory system that, while some asymmetry exists at birth, requires extensive and challenging binaural listening experiences leading up to adulthood to ensure the full maturation of the asymmetry and the associated speech-in-noise recognition capabilities.\n\nOnly when the depth of our knowledge of the central auditory system (CAS) matches that of the peripheral auditory system, and audiological practice is informed by the integration of this knowledge, can we expect to effectively rehabilitate those with hearing problems. While our knowledge of the CAS remains far from complete and this study and model make only a minor contribution in this respect, one clear conclusion has been arrived at—auditory science is compromised by a lack of “asymmetry sensitive” tools.\n\nWe have hitherto thought of hearing as nature. We now know that plasticity is a defining feature of the auditory system during its maturation, middle age, and the years of presbycusis. We need to think more seriously about how best the auditory system can be nurtured.\n\n\nData availability\n\nNo data is associated with this article.", "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\nAndersson B, Hugdahl K: Effects of sex age, and forced attention on dichotic listening in children: A longitudinal study. Dev Neuropsychol. 1987; 3(3–4): 191–206. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "35211", "date": "28 Jun 2018", "name": "Vasiliki Iliadou", "expertise": [ "Reviewer Expertise Auditory Processing 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 very interesting and well-written article: “Life experience and the asymmetry of the human auditory system/ clinical and auditory science laboratory implications” taps into a very important issue. Laterality of the auditory system although widely recognised is not yet fully implemented in clinical evaluation in everyday audiological practice. This leads to less than optimal management of hearing impairment as this is not restricted to hearing loss but extends to auditory processing disorder. The author should more clearly include auditory processing disorder (APD/CAPD) and its test battery approach which includes dichotic listening. Especially since APD evaluation is the only case in clinical audiology where asymmetry of the auditory system is measured, accounted for and remediated. Dichotic listening tests are at the core of APD test battery that lead to APD diagnosis. The author should refrain from using the term “suspected APD” as this is misleading and is not a diagnostic label. The term APD should be used referring to properly evaluated and diagnosed individuals.\n\nBoth abstract and keywords should include Auditory Processing Disorder as it is during an auditory processing evaluation that asymmetries in the HAS are studied and revealed Shen present. For example, the right ear advantage during a dichotic test evaluation may reveal typical development and the absence or reversal of asymmetry may indicate an individual with pathological evidence concerning the central auditory nervous system.\nIntroduction is very well written and it should lead up to how important clinical evaluation of auditory processing is to be able to better describe, evaluate and rehabilitate speech in noise difficulties for example.\nThe dichotic listening test: The text provides a good description of dichotic listening tests with different stimuli and conditions. It lacks the inclusion and description of clinical populations. To be able to tap into hearing as a whole and not focus on the peripheral hearing alone, clinical populations and their results showing how asymmetry is presented or if it is still present should be discussed and compared with that of normal populations. The latter is very well presented but clinical population results should be included in this section.\nThe corticofugal modulation of the asymmetrical auditory system: In this section the author writes: “The Bergen Dichotic Listening Database [Westerhausen et al., 2015] and the resulting normative data provided with their test manual [Hugdahl & Asbjørnsen, 2013] enables an understanding of the effects of age upon the REA-CV. There has been little discussion of these age effects in the literature but they provide valuable insight into the lengthy maturity window of the human auditory system through childhood and adolescence, and also its gradual decline, beginning as early as the sixth decade.” These databases however do not usually include the hearing threshold that represents the state of the peripheral hearing. If the author has these data he should present them and so how even though loss of hearing sensitivity is present with the ageing process, this can be clearly separated from the differences in dichotic listening results in younger and older adults. In the case that data of the pure tone audiometry (hearing threshold across tested frequencies) are not present the author should rephrase this section to include the possibility that the results showing in the dichotic listening may be the result of presbyacusis.\nAC asymmetry: Please spell out AC. What does it stand for?\nThe author states that: “The Bergen normative data [Hugdahl & Asbjørnsen, 2013] shows that there is a large between-individual variability.” Can he really rule out that this may be due to different hearing thresholds (peripheral hearing)?\nIt would add to the value of this paper to include results of studies regarding medial olivocochlear system functionality (otoacoustic emissions suppression). In these studies there is a documented asymmetry as well that is part of the HAS and it should be included. Examples of such studies are:\n\nIliadou, V. V., Weihing, J., Chermak, G. D., & Bamiou, D. E. Otoacoustic emission suppression in children diagnosed with central auditory processing disorder and speech in noise perception deficits1. Section 4.3. is discussing “Mechanism for laterality differences”.\nS. Khalfa, L. Collet Functional asymmetry of medial olivocochlear system in humans. Towards a peripheral auditory lateralization Neuroreport2.\n\nE. Veuillet, A. Magnan, J. Ecalle, et al. Auditory processing disorder in children with reading disabilities: effect of audiovisual training3.\n\nS.G.G. Sanches, R.M. Carvallo Contralateral suppression of transient evoked otoacoustic emissions in children with auditory processing disorder4.\nThe author writes that: “The vast majority of the speech-in-noise studies in the literature describe their use of speech audiometry tests that use headphones. Such tests do not represent realistic listening situations, and do not test speech-in-noise skills that relate to the asymmetry of the auditory system”. Although this may be partly true, it should be made clear that the reason behind this testing methodology is to ensure that even in cases when the testing environment does not permit for ideal acoustic conditions (sound treated booths instead of soundproof ones, for example or a quiet room vs a sound treated booth) testing is accurate. This should be included to balance the information provided in this section.\nIn the section: “Childhood development and the in-built asymmetry of the ACs” the author should rephrase results concerning OAE suppression providing insight into laterality/asymmetry. Please see paper by Gkoritsa et al 2007 indicating that (i) MOCB maturation is thought to be complete before the age of five in children and (ii) the presence of suppression laterality indicates full maturation5.\n\nIn discussion (last sentence of the second paragraph) the author states that: “In sum, while some asymmetry exists at birth, it is life experience that shapes the detail of the auditory processing capability and the functional asymmetry of an individual's auditory system.”\n\nLife experience with an intact and fully functioning peripheral and central auditory system should be added to stress the fact that this will be altered if a hearing loss or an auditory processing disorder is present.\n\nIn the section: “Using the model of the asymmetrical auditory system” auditory processing test battery approach that is used in specialised APD/CAPD clinics throughout the world should be mentioned and described. See references in6.\nIn the section “Extending the model of the asymmetrical auditory system” the phrase “The management of those suspected to have Auditory Processing Disorder” SHOULD change to “The management of those diagnosed with Auditory Processing Disorder” and the reference of Moore et al, 2018 should be omitted as not relevant. It is not acceptable to mix populations together and it is not acceptable to manage suspected and not diagnosed individuals. We do not manage suspected hearing loss for example, we have to first diagnose it to know how to manage it. The same is true for APD. Include references:\n\nIliadou V and Kiese-Himmel C. Common Misconceptions Regarding Pediatric Auditory Processing Disorder7. Sharma M, Purdy SC, Kelly AS. A randomized control trial of interventions in school-aged children with auditory processing disorders8 Iliadou V, Sirimanna T, Bamiou DE. CAPD is classified in ICD-10 as H93.25 and hearing evaluation-not screening-should be implemented in children with verified communication and/or listening deficits9. Musiek FE, Chermak GD, Weihing J, Zappulla M, Nagle S. Diagnostic accuracy of established central auditory processing test batteries in patients with documented brain lesions10.\nThe section on Hidden hearing loss should be preceded by one on APD. See Musiek, F. E., Chermak, G. D., Bamiou, D. -., & Shinn, J. (2018)11. CAPD: The most common ‘hidden hearing loss’ central auditory processing disorder-and not cochlear synaptopathy-is the most likely source of difficulty understanding speech in noise (despite normal audiograms).\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? 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": [] }, { "id": "35836", "date": "26 Jul 2018", "name": "Stefan Bleeck", "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 written piece that in my opinion is important and justified to index. The author makes the argument that current clinical practice does not recognise asymmetries in the central auditory system that might be responsible for individual differences in speech in noise perception. The author gives a critical literature review about research in the asymmetric auditory system with regards to structure, function, development and modelling. This is an excellent review in this area that has been lacking so far. The authors conclusion is that the observed asymmetry could be (partially) also responsible for observed individual differences in speech in noise performance. This connection has – to my knowledge – never been made and is an interesting suggestion. There is certainly not enough research in this area (as the author points out) and the question of causal relationships needs deeper investigation, but the conclusion that today’s clinical speech tests that are performed mostly unilaterally are difficult to interpret is (in my opinion) absolutely justified. The author concludes with recommendation for clinical work that are straightforward and should be considered: more use of high frequency audiometry (which is not related to the topic in this paper, but absolutely justified from existing literature), and more performance of free field speech audiometry (before and after fitting).\nA few points could improve the paper further: The superior olives and the inferior colliculus are generally thought to be the first fully bilateral sound processing stations in the auditory pathways and many authors suspect that they are involved in the speech of noise task. To my knowledge there has never been an asymmetry been observed (or even suspected) in the brainstem processing. However, from there, the auditory streams go to either auditory cortex. So, it is not quite the case that (as implied in figure 1), the information stream goes straight from the ears to the contralateral cortex. I don’t immediately see how this affects the main argument of AC asymmetry, but I feel for completeness that this should be discussed. Many authors don’t give the brainstem the due recognition in the role for speech understanding – understandable as there is little research in this area.\nThe Bergen experiment is central and explanation takes up a lot of space, however, it is not explained well. I needed to reread the page several times before I understood what the bars in figure 3 are. Also: where is the data from? Error bars? What does ‘contrived’ mean in the legend. Please rephrase the legend so that this interesting experiment becomes understandable to the non-expert reader.\nSame with figure 4: where is the data from? Re-worked? What are the error bars? You are talking about the inter-individual difference later, but it’s not clear here how large the effect is in relationship to their variance. The paragraph about the hidden hearing loss actually doesn’t add anything to the discussion about asymmetry or did I miss that? It’s all correct, but what is the point of it for this paper, other than giving a second clinical recommendation (as much as I support it!)\n\nIn conclusion, I really enjoyed reading this and am planning some undergrad experiment later this year to find out more about the asymmetry of speech in noise recognition.\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/7-675
https://f1000research.com/articles/7-298/v1	08 Mar 18	{ "type": "Research Note", "title": "Using electronic biology based platform to predict flu vaccine efficacy for 2018/2019", "authors": [ "Slobodan Paessler", "Veljko Veljkovic", "Slobodan Paessler" ], "abstract": "Flu epidemics and potential pandemics pose great challenges to public health institutions, scientists and vaccine producers. Creating right vaccine composition for different parts of the world is not trivial and has been historically very problematic. This often resulted in decrease in vaccinations and reduced trust in public health officials. To improve future protection of population against flu we urgently need new methods for vaccine efficacy prediction and vaccine virus selection.", "keywords": [ "influenza", "vaccine efficacy", "H3N2", "electronic biology" ], "content": "Introduction\n\nVaccine effectiveness (VE) against H3N2 viruses is typically lower than VE against influenza H1N1 and/or influenza B viruses. It's not uncommon to see VE of about 30 percent against H3N2 viruses. Furthermore, during the flu season 2017 in Australia, VE of the seasonal flu vaccine was around 10% resulting in record-high numbers of laboratory-confirmed influenza A infections, hospitalizations and deaths1. This situation raised concerns that similar could happen in the United States during the flu season 2017/2018, in which H3N2 viruses were predominant. The concerns were based on assumptions that H3N2 viruses in Australia and US were similar, as the classical phylogeny indicated, and because the vaccine composition was identical one could expect comparable levels of VE. Therefore, predicted VE of the flu vaccine in the USA at the beginning of the flu season was around 10%2. This prediction was justified and rationalized using the assumption that H3N2 viruses circulating in Australia in the flu season 2016–17 are similar to viruses in the Northern Hemisphere.\n\nComparison of Australian H3N2 viruses and viruses isolated in the USA at the beginning of the flu season 2017–18, performed using a novel functional phylogenetic tool, demonstrated significant difference between these two groups of viruses3. This new information led us to predict that the flu vaccine in US should work in the season 2017–18 just as well as in 2016–173. Our prediction was recently confirmed in the interim CDC estimation of 2017–18 seasonal influenza VE, published and released in February 20181. Moreover, the risk for a (H3N2) associated medically-attended influenza illness was reduced through vaccination by 59% among children aged 6 months through 8 years1.\n\n\nMethods\n\nTo improve VE for the flu season 2018, WHO selected in September 2017 the new vaccine virus A/Singapore/INFIMH-16-0019/2016, which is better adapted to H3N2 viruses circulating in the South Hemisphere (See WHO recommendation of vaccine compositions for the Southern Hemisphere). The WHO in February 2018 selected the same virus for the vaccine for the season 2018–19 in the North Hemisphere (See WHO recommendation of vaccine compositions for the Northern Hemisphere).\n\nIn order to assess VE against H3N2 viruses for the next flu season 2018–19 in the United States, we analyzed compatibility between new vaccine virus A/Singapore/INFIMH-16-0019/2016 and H3N2 viruses isolated in 2018 in US. This analysis was performed using the informational spectrum method (ISM) based phylogenetic algorithm, the Informational Spectrum-based Phylogenetic Analysis (ISTREE), which we previously used to assess VE for the flu season 2017–183. This algorithm, which is based on the informational hallmark of proteins that determines their biological function, was previously described in more detail4.\n\n\nResults and discussion\n\nIn Figure 1 the ISM-based phylogenetic tree is presented for hemagglutinin HA1 from 68 H3N2 viruses collected in the United States from January to February 2018 and stored in the publicly open database GISAID. As can be seen in this figure, the H3N2 viruses are grouped into two separate clusters and the novel vaccine virus A/Singapore/INFIMH-16-0019/2016 belongs to the small cluster encompassing only 8.8% of analyzed viruses. Previously we showed that 71% of H3N2 viruses isolated in the beginning of the US season 2017–18 were informationally compatible with vaccine virus3. This compatibility resulted in good protection against H3N2 viruses in this season1. The low informational compatibility between new vaccine virus and H3N2 viruses circulating in US suggests that VE for the next flu season in US could be very low. Of note is that H3N2 virus A/Hong Kong/4801/2014 in vaccine for the season 2017–2018 better fits US viruses than new vaccine virus A/Singapore/INFIMH-16-0019/2016. This suggests possibility that VE of the current vaccine could be even higher than that for the new vaccine.\n\nThe vaccine viruses are marked with asterisk (green).\n\nWe propose the “ISM-based phylogenetic algorithm ISTREE analysis” for rapid and accurate analysis of different influenza A viruses that can be used for VE prediction. This is a first report VE prediction prior to flu season using computational analysis. Our prediction has been recently confirmed through laboratory reports released by CDC. Based on current data, we predict low VE for the season 2018/2019 for US due to vaccine virus selection.\n\n\nData availability\n\nSequence data of the viruses were obtained from the GISAID EpiFluTM 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 EpiFluTM Database and Data are defined by the Terms of Use.\n\nDataset 1: Human H3N2 influenza viruses collected in the United States from January to February 2018 (GISAID EpiFluTM database, accessed February 20, 2018). 10.5256/f1000research.14140.d1962235", "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\nFlannery B, Chung JR, Belongia EA, et al.: Interim estimates of 2017-18 seasonal influenza vaccine effectiveness - United States, February 2018. MMWR Morb Mortal Wkly Rep. 2018; 67(6): 180–185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaules CI, Sullivan SG, Subbarao K, et al.: Chasing Seasonal Influenza - The Need for a Universal Influenza Vaccine. N Engl J Med. 2018; 378(1): 7–9. PubMed Abstract \| Publisher Full Text\n\nPaessler S, Veljkovic V: Prediction of influenza vaccine effectiveness for the influenza season 2017/18 in the US [version 1; referees: 2 approved]. F1000Res. 2017; 6: 2067–2075. Publisher 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(8): 2238–2255. Publisher Full Text\n\nPaessler S, Veljkovic V: Dataset 1 in: Using electronic biology based platform to predict flu vaccine efficacy for 2018/2019. F1000Research. 2018. Data Source" }	[ { "id": "31711", "date": "22 Mar 2018", "name": "Daniel R. Perez", "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 provocative research article based on the authors' previous development of the informational spectrum method that takes into account how virus/receptor interactions modulate the antigenic cross-reactive phenotype of the HA protein of influenza A viruses. Using this method, the authors were able to shed light on the lack of efficacy of the vaccine against the H3 subtype during the 2014/15. They were also able to predict that in the US, the vaccine against the H3 subtype was going to be more efficacious than previously anticipated by the CDC. Later in the 2017/18 season, the CDC confirmed the authors' prediction. In this latest article, the authors predict that the selection of the vaccine candidate for the 2018/19 is less than ideal and suggest that vaccine efficacy against the H3 subtype will be lower than in this past season. This type of work is fascinating and necessary and hopefully it becomes part of the toolkit for the selection of vaccine candidates against influenza.\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": "31709", "date": "22 Mar 2018", "name": "Abdel-Satar Arafa", "expertise": [ "Reviewer Expertise Virology", "avian influenza", "genetic 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 article “Using electronic biology based platform to predict flu vaccine efficacy for 2018/2019” is clearly presented and technically sound enough for publication in F1000Research online. It is well-written and supported by computational well-developed bioinformatics analysis.\nI just suggest one observations that should be clarified to enhance the work: -\n\nFig 1 The ISM tree based analysis is not supported by a tool to predict grouping like bootstrapping method to confirm accurate grouping\n\nFinally, the article is recommended for publication in its present format.\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": "31708", "date": "27 Mar 2018", "name": "Timm C. Harder", "expertise": [ "Reviewer Expertise Virology", "diagnostics", "and epidemiology of influenza A" ], "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 provide an analysis of HA sequences of recent H3N2 influenza viruses from the ongoing season using the protein sequence-based clustering algorithm ISTREE. This method has been developed and published by one of the authors several years ago. ISTREE seeks to combine molecular data and provide a clustering with respect to antigenic relatedness of the influenza virus HA. Previous publications provided a retrospective analysis of genetic and antigenic data. Basically, ISTREE is advertised as an alternative to antigenic cartography of influenza viruses using serologic data based on hemagglutination inhibition (HI).\nIn fact, such algorithms would be of great interest and value in terms of vaccine selection. The prospective statements re appropriate vaccine strains for a future influenza season made by the authors here are quite clear but they are solely based on ISTREE assessments. No flanking/supporting data based on standard HI assays is provided. Assessing the validity of the ISTREE data therefore is not possible. The data may well turn out to be useful but, as it currently stands, they may be totally misleading as well.\nSolid antigenic data will be required to validate the potentially useful ISTREE algorithm.\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? 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": "3576", "date": "04 Apr 2018", "name": "Veljko Veljkovic", "role": "Author Response", "response": "The major Referee’s criticism concerning our study is the lack of serology/virology data to support our approach for making valid predictions. This criticism misses the major point of the study, which is to rapidly release predictions based on in silico screening to allow other scientists to look at it as early as possible and to test it through the flu season. However, as we have mentioned in our paper we used this method previously and if the reviewer would have read the paper carefully, he would have realized that our prediction for the flu season 2017/18 was confirmed several months later by CDC using various serological assays. These data can be found in CDC laboratory reports (see the latest CDC report: https://www.cdc.gov/flu/weekly/index.htm) and present a totally independent validation of our approach." } ] } ]	1	https://f1000research.com/articles/7-298
https://f1000research.com/articles/7-673/v1	29 May 18	{ "type": "Software Tool Article", "title": "A web resource for nutrient use efficiency-related genes, quantitative trait loci and microRNAs in important cereals and model plants", "authors": [ "Anuj Kumar", "Ajay Pandeya", "Girik Malik", "Mansi Sharma", "Hima Kumari P.", "Anil Kumar S.", "Vijay Gahlaut", "M.N.V. Prasad Gajula", "Krishna Pal Singh", "Prashanth Suravajhala", "Harindra Singh Balyan", "Pushpendra K. Gupta", "Anuj Kumar", "Ajay Pandeya", "Girik Malik", "Mansi Sharma", "Hima Kumari P.", "Anil Kumar S.", "Vijay Gahlaut", "M.N.V. Prasad Gajula", "Krishna Pal Singh" ], "abstract": "Cereals are key contributors to global food security. Genes involved in the uptake (transport), assimilation and utilization of macro- and micronutrients are responsible for the presence of these nutrients in grain and straw. Although many genomic databases for cereals are available, there is currently no cohesive web resource of manually curated nutrient use efficiency (NtUE)-related genes and quantitative trait loci (QTLs). In this study, we present a web-resource containing information on NtUE-related genes/QTLs and the corresponding available microRNAs for some of these genes in four major cereal crops (wheat (Triticum aestivum), rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare)), two alien species related to wheat (Triticum urartu and Aegilops tauschii), and two model species (Brachypodium distachyon and Arabidopsis thaliana). Gene annotations integrated in the current web resource were manually curated from the existing databases and the available literature. The primary goal of developing this web resource is to provide descriptions of the NtUE-related genes and their functional annotation. MicroRNAs targeting some of the NtUE related genes and the QTLs for NtUE-related traits are also included. The genomic information embedded in the web resource should help users to search for the desired information.", "keywords": [ "Nutrient use efficiency", "cereals", "web-resource", "functional genomics", "genes", "QTLs", "miRNAs", "stress" ], "content": "Introduction\n\nThe world population is expected to be >9.6 billion by 2050. As a consequence, food production must increase by 70% to meet the growing demand for food; this translates into 3 billion tons of additional cereal grain1. In view of this, food and nutritional security have been central to several international discussions, involving development of strategies for enhanced and sustainable food production2. These strategies include the development of cultivars that would be (i) resilient to climate change, (ii) tolerant to biotic and abiotic stresses and (iii) efficient to the use of fertilizer and water resources. In connection with strategy (iii), it is well known that the present day high-yielding cultivars of a majority of crops have a high demand for inputs including water and nutrients.\n\nOut of the 17 essential elements needed for plant growth and development, three non-mineral elements [C (carbon), H (hydrogen) and O (oxygen)] are derived by the plants from CO2 and H2O, and the remaining 14 mineral elements are ordinarily derived from the soil as inorganic salts. These mineral elements include: (i) six macronutrients [N (nitrogen), P (phosphorous), K (potassium), S (sulphur), Ca (calcium) and Mg (magnesium)] that are required in large quantities (1,000–15,000 mg/kg of plant dry weight); and (ii) eight micronutrients [B (boron), Cl (chlorine), Cu (copper), Fe (iron), Mn (manganese), Mb (molybdenum), Zn (zinc) and Ni (nickel)] that are needed in relatively small quantities (0.1–100 mg/kg of plant dry weight)3.\n\nSince most soils are deficient in N, P and K, these elements are applied to the crops in the form of chemical fertilizers to help their proper growth and development, eventually leading to improved grain yield4,5. Of these three macronutrients, N and P are required in the maximum quantities by the plants6. With the increased future demand for cereal grains, especially within developing countries, the demand for N and P is likely to grow7.\n\nDuring the last four decades, the doubling of food production worldwide has been associated with 7-fold increase in the consumption of N fertilizers and 3.5-fold increases in the consumption of P fertilizers8,9. It is estimated that a further doubling of global food production during the next three decades would require a 3.15-fold increase in total N application and 2.5-fold increase in total P application8. The enhanced use of chemical fertilizers would also add up to 50% (for N fertilizers) of the operational costs in agriculture4.\n\nIt is estimated that 30–50% of N applied in the fields is used by the crops10, with the excess N and ammonia converted by the soil bacteria into nitrate and nitrite, which are lost via emission in the form of NH3, N2O and NO2 gases, and/or leached out into the soil in the form of nitrate ions (NO3−)4,11, causing eutrophication in terrestrial and aquatic systems. N2O is 300 times more potent as a greenhouse gas (GHG) than CO2; 70% of N2O is contributed by natural sources and is responsible for considerable GHG effects, which have a significant influence on climate change7,12,13.\n\nUnlike N, part of the P derived from applied chemical fertilizers is held very tightly by the surface of the soil particles or is fixed as organic P compounds, thus remaining unavailable to the plant for uptake; P is also lost through leaching into the ground and surface waters, damaging the surrounding environment14. The leached P also leads to eutrophication and associated algal blooms, decreased dissolved O2, foul odour, and generally poor water quality15. To further exacerbate the problem, the P used in fertilizers is obtained from rock phosphate, which is a non-renewable source and should be depleted in the next 50–100 years16. Indirectly through the food chain, application of enhanced N and P fertilizer also leads to widespread deficiency of micronutrients in human population, thus making it a health hazard.\n\nIn view of the importance of nutrient use efficiency (NtUE) as an agronomic practice in crop breeding, the European Commission recently concluded a 5-year research project 'NUE-CROPS' with the aim of improving NtUE in four major European food, feed and biofuel crops to reduce the negative environmental impact of crop production (EU-FP7 222-645). The findings of this project were discussed at a EUCARPIA section meeting on 'Organic and Low-input Agriculture' during September 24–26, 2013 at the George August University of Gottingen and the proceedings were published in Euphytica17.\n\nTraditional breeding practices involving higher application of chemical fertilizers are no longer providing yield improvements18. The Food and Agriculture Organization of the UN has noticed that since the mid-1980s, crops, including wheat, maize and soybean, have shown only 1% annual growth in their productivity, whereas in developed countries the annual growth in crop yield has remained static, with no improvement in productivity19. Thus it has been a challenge for the geneticists and plant breeders to achieve the desired increase in the productivity of cereal crops in particular, and that of the other food crops in general, without compromising the quality of food and environment health. Of the suggested strategies, the development of NtUE crop plants through genetic intervention is the best approach, which is also environment-friendly. This approach would require reduced levels of fertilizer application, with the desired increase in current grain yield levekls7,9,14,19. Also termed \"An Evergreen Revolution\" and the “Second Green Revolution”, this approach would allow increased productivity, while following sustainable agricultural practices20,21.\n\nAn increased understanding of the NtUE at the genetic and molecular levels would certainly help in developing NtUE cultivars giving enhanced crop productivity. The NtUE (ratio of grain yield to units of nutrient supplied or available) can be divided in two components: (i) nutrient uptake efficiency (the ratio of total plant nutrients to the available or supplied nutrient), which is the ability of the crop plant to extract nutrients from the soil; and (ii) nutrient utilization efficiency (the ratio of grain yield to total plant nutrients in grain and straw), which measures the capacity of the plant to convert the absorbed nutrient into grain yield22,23. Another approach is to calculate the above-ground biomass NtUE, which is measured by dividing the dry shoot weight by the nutrient supplied24; however, plant breeders are most often interested in the first approach of measuring the NtUE in breeding of grain crops.\n\nThe NtUE is a complex trait, involving signaling, acquisition, transport and utilization, the latter constituting assimilation and translocation/remobilization25. The detailed description of the mechanisms of signaling, uptake, transport and utilization of the nutrients is neither necessary nor desirable in this article, but we know that they form an interwoven network of genetic control. Thus, the identification of genes/QTLs and the microRNAs (miRNAs) targeting the NtUE-related genes involved in the uptake, transport and utilization of nutrients in different parts of the plant are important targets for breeding cultivars with improved NtUE. The genes that have been identified or are likely to be identified in future, or the DNA markers associated with these genes/QTLs as well as the information related to miRNA can be exploited as follows: (i) marker-assisted selection aimed at the selection of desirable genotypes exhibiting improved plant performance under reduced nutrient supply; (ii) a study of allelic variation of the genes/QTLs for their subsequent exploitation in plant breeding, and (iii) the development of transgenics with improved NtUE25,26.\n\nA number of genomic resources are freely available for cereals and include the following: Gramene27, PIGD28, PlantTFDB 43.029, PSPDB30, CerealsDB31, MetaCrop 2.032, Phytozome33, PlantGDB34, CSR-DB35, CR-EST36 and GrainGenes37. However, a similar web resource for NtUE genes or QTLs is not available, although the USDA has developed a tool for calculating the approximate amounts of nutrients (N, P and K) required for an optimum harvest of each agricultural crop. Therefore, we have developed the NtUE web-resource, which provides comprehensive information on genes related to the components of NtUE that are involved in processes such as the uptake, transport, nitrate assimilation, re-assimilation, amino acid biosynthesis, C/N storage and metabolism, signaling and regulation, translocation, remobilization, senescence, regulation, DNA-binding, ion-binding and copper homeostasis. The web resource also contains information on QTLs for different NtUE-related traits, such as shoot growth, total N, nitrate, free-amino acid contents, relative grain yield, relative biomass yield, relative grain N, relative biomass N, N response, leaf length, P starvation, and Fe and Zn homeostasis. The miRNAs targeting some of the NtUE-related genes are also included in the web resource. The NtUE web resource is, to our knowledge, the first of its kind. We believe that the web resource will be useful for members of the plant research community, especially plant breeders, who may like to plan experiments related to the improvement of NtUE of cereal crop plants.\n\n\nMethods\n\nWe populated the entries in the web resource by retrieving NtUE-related genes using gene ontology keyword searches for nutrients, plants, crops and their NtUE-related role from GenBank, EnsemblPlants, Gramene and UniProt38. We also extensively searched PubMed with different keywords or their combination to find relevant articles.\n\nWe performed functional annotation studies at gene and protein level to develop comprehensive information for the collected NtUE responsive genes. Complementary- and target-site accessibility both are important factors of plant regulatory small RNA target recognition mechanism39. To predict potential miRNAs targeting the NtUE responsive genes in plant species, the web-based psRNATarget server40 was used with the default parameters. Further the QTLs for NtUE related traits were manually curated from the available scientific literatures, GrainGenes database and MaizeGDB.\n\nThe NtUE web resource was set up based on three-tier architecture concept using Apache/PHP/MySQL on a Windows 8.1 platform. An integrated system driven through MySQL (5.6.21) and PHP (5.6.24) was developed to handle the storage of annotated 688 genes in the web resource. The database can be accessed from any operating system client recommended with Java and internet connection. A flow-chart depicting the steps involved in preparation of web resource and acquisition of data by the user is presented in Figure 1. All figures have been drawn using MS office tools.\n\n\nUse of NtUE web resource\n\nNtUE is a comprehensive web resource containing information on NtUE-related genes/QTLs and the microRNAs targeting some of these genes in three major cereal crops (wheat (T. aestivum), rice (O. sativa), maize (Z. mays)), two alien species (T. urartu and A. tauschii) related to wheat, and two model species (B. distachyon and A. thaliana).\n\nIn the current release, we have compiled 688 NtUE-responsive gene records that were manually curated and annotated using different bioinformatics-based methods. Each entry embedded in this web resource has been arranged in a gene-centric manner for easy access and retrieval.\n\nEach entry embedded in the web resource provides the following comprehensive information on NtUE-responsive genes: (i) organism name; (ii) type of associated nutrient (macronutrients and micronutrients); (iii) gene symbol; (iv) gene ID (entry accession on UniProt, GenBank, Gramene and EnsemblPlants), with an active hyperlink that provides genomic information and sequences of entry on portal of parental database; (v) gene description; (vi) function; (vii) linked miRNAs with an active hyperlink to miRBase database; (viii) putative QTLs; (ix) chromosomal location marker; (x) information on experimental condition of the trait; and (xi) references for QTLs (as shown in Figure 2).\n\nThe information available in the web resource could be accessed through keyword search tab by assigning the nutrient name (e.g. N, P, K, Zn, Fe), organism name (e.g. Arabidopsis thaliana, Brachypodium distachyon, Oryza sativa, Triticum aestivum, Hordeum vulgare), gene name (NRT1.1, PHT1.1), accession number and clicking on the “search” button. After submitting the keyword, a page will open presenting a list of NtUE-responsive gene details, which contains the specific function of the gene. Clicking on “MORE INFORMATION” will direct the user to entire information for a particular gene. This scroll down based page provides comprehensive information that includes a summary of gene including: organism name, type of nutrient (macronutrients and micronutrients), gene information (gene symbol, gene ID, gene description, function) target miRNAs, and QTLs (chromosomal location marker, information on experimental condition of the trait and references for QTLs). Alternatively, the user can search the NtUE web resource using the advanced search option, which will allow them to conduct an organism-specific search using a gene name (e.g. NRT1.1, PHT1.1) or accession number. Using a specific example of search option, the steps involved in using the keyword-based search and advanced search options are shown in Figure 3. The NtUE web resource has a user-friendly entry point for each gene/miRNA/QTL. Each gene with a corresponding miRNA and the record of the corresponding QTL reference article in the web resource is linked to external resources, including NCBI, UniProt, Gramene, EnsemblPlants and miRBase. Using external links, users can get detailed genomic and proteomic information on NtUE-associated genes, miRNA-target genes and the trait for each QTL. Additionally, it was ensured that throughout, standard terminology is used, validated and processed, making the tool an easily understandable tool for accessing genomic information using NtUE. QTLs embedded in the NtUE web resource contain the chromosomal location, marker interval, trait information and reference article, with an external link to the journal in which the article is published. Figure 3 shows an example of how more information on genes and targeted miRNAs can be retrieved.\n\nType of nutrients. At present, the NtUE web resource covers 668 NtUE-responsive genes, corresponding to 12 nutrient types (the nutrient-wise distribution of NtUE genes in the NtUE web resource is shown in Figure 4). It is apparent from the graph that the majority of NtUE genes are associated with N. The other major nutrients embedded in the web resource for which NtUE genes have been detected are K, P, Cu and Zn.\n\nNtUE-responsive genes in cereals and model plant species. NtUE-responsive genes have been reported in literature published in peer-reviewed journals. These genes have been detected from the genomes of eight model and cereal plant species (embedded in the web resource), including Arabidopsis, rice, wheat and maize (Figure 5). Among all the embedded plant genomes, Arabidopsis are the best-explored source of annotated NtUE-responsive genes in the NtUE web resource.\n\nComparison of NtUE web resource with Gramene. Gramene is a biological repository that provides genomic information about members of the Poaceae family. Gramene is also an experimentally based comprehensive database consisting of genomic sequences, chromosomal locations, associated QTLs and markers for genes from various popular genomes belonging to the Poaceae family. In contrast, the NtUE web resource is a literature-curated resource for NtUE-responsive genes of model-organisms and cereals; these genes differ for different nutrients and in different plant genomes. The NtUE web resource not only lists the NtUE genes according to their nutrient type, but also provides data on aspects such as miRNA targets, associated QTLs, marker associated with QTL, information on trait and experiment and external links to literature on QTL. Only NtUE genes from cereals overlap with those in Gramene, but the NtUE genes from model plant species are unique to the NtUE web resource.\n\n\nConclusions\n\nThe NtUE web resource is, to our knowledge, the first of its kind to provide a comprehensive non-redundant catalogue of NtUE genes that have a vital role in model and cereal plant species during plant growth and development, with supporting evidence from published literature. We hope that this web resource will be a useful compendium to the geneticists, plant breeders and computational biologists involved in crop improvement. The resource contains manually curated entries with an adequate level of stringency to select only those genes/QTLs/miRNAs that are validated and linked to NtUE. This resource will help in providing new solutions to plant geneticists and breeders who are aiming towards the sustainable use of limited soil nutrients for enhanced productivity without compromising the nutritional quality of the grains. Information on more than half of these genes, QTLs and miRNAs is not available in the previously generated databases. With further progress in the identification of genes/QTLs/miRNAs for NtUE related traits, we anticipate that these numbers will eventually grow by two- to three-fold.\n\n\nData and software availability\n\nThe dataset for NtUE web-resource is publically available on OMIC TOOLS with unique identifier OMICS_24069.\n\nThe archived PHP files for the web resource at the time of publication have been deposited at Zenodo: https://doi.org/10.5281/zenodo.123301141.\n\nLicense: Creative Commons Attribution 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\nWe thank Dr. V.S. Sundararajan for his useful suggestions on the prototype of the database. We gratefully acknowledge Prof. P.B. Kavi Kishor for proofreading the manuscript.\n\n\nReferences\n\nLafiandra D, Riccardi G, Shewry PR: Improving cereal grain carbohydrates for diet and health. J Cereal Sci. 2014; 59(3): 312–326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEpstein A: Mineral metabolism. In: Plant Biochemistry. (J Borner and JE Varner, eds). Academic Press, London. 1965; 438–466.\n\nHirel B, Tétu T, Lea PJ, et al.: Improving nitrogen use efficiency in crops for sustainable agriculture. Sustainability. 2011; 3(9): 1452–1485. Publisher Full Text\n\nAlvarez JM, Vidal EA, Gutiérrez RA: Integration of local and systemic signaling pathways for plant N responses. Curr Opin Plant Biol. 2012; 15(2): 185–191. PubMed Abstract \| Publisher Full Text\n\nEpstein E, Bloom A: Mineral nutrition of plants: principles and perspectives. Sinauer Associates, USA. 2005. Reference Source\n\nGood AG, Beatty PH: Biotechnological Approaches to Improving Nitrogen Use Efficiency in Plants: Alanine Aminotransferase as a Case Study. The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops. John Wiley & Sons, Inc., Sussex. 2011; 165–192. Publisher Full Text\n\nTilman D: Global environmental impacts of agricultural expansion: The need for sustainable and efficient practices. Proc Natl Acad Sci U S A. 1999; 96(11): 5995–6000. PubMed Abstract \| Publisher Full Text\n\nHirel B, Le Gouis J, Ney B, et al.: The challenge of improving nitrogen use efﬁciency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. J Exp Bot. 2007; 58(9): 2369–2387. PubMed Abstract \| Publisher Full Text\n\nGarnett T, Conn V, Kaiser BN: Root based approaches to improving nitrogen use efficiency in plants. Plant Cell Environ. 2009; 32(9): 1272–1283. PubMed Abstract \| Publisher Full Text\n\nSchlesinger WH: Biogeochemistry: An Analysis of Global Change. Academic, San Diego. 1991. Reference Source\n\nWuebbles DJ: Atmosphere. Nitrous oxide: no laughing matter. Science. 2009; 326(5949): 56–57. PubMed Abstract \| Publisher Full Text\n\nKochian LV: Plant nutrition: Rooting for more phosphorus. Nature. 2012; 488(7412): 466–467. PubMed Abstract \| Publisher Full Text\n\nReynolds MP, Quilligan E, Aggarwal K, et al.: An integrated approach to maintaining cereal productivity under climate change. Glob Food Sec. 2016; 8: 9–18. Publisher Full Text\n\nCorrell DL: The role of phosphorus in the eutrophication of receiving waters: A review. J Enviorn Qual. 1998; 27(2): 261–266. Publisher Full Text\n\nCordell D, Drangetra JO, White S: The story of phosphorus: Global food security and food for thought. Global Env Chang. 2009; 19(2): 292–305. Publisher Full Text\n\nLammerts Van Bueren E, Thorup-Kristensen K, Leifert C, et al.: Breeding for nitrogen efficiency: concepts, methods, and case studies. Euphytica. 2014; 199(1–2): 1–2. Publisher Full Text\n\nFischer RA, Byerlee D, Edmeades GO: Can technology deliver on yield challenge to 2050? FAO Expert Meeting on How to Feed the World in 2050. 2009; 24–26. Reference Source\n\nGamuyao R, Chin JH, Pariaska-Tanaka J, et al.: The protein kinase Pstol1 from traditional rice confers tolerance of phosphorus deficiency. Nature. 2012; 488(7412): 535–539. PubMed Abstract \| Publisher Full Text\n\nSwaminathan MS: An evergreen revolution. Crop Sci. 2006; 46(5): 2293–2303. Publisher Full Text\n\nZeigler RS, Mohanty S: Support for international agricultural research: current status and future challenges. N Biotechnol. 2010; 27(5): 565–572. PubMed Abstract \| Publisher Full Text\n\nMoll RH, Kamprath EJ, Jackson WA: Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization. Agron J. 1982; 74(3): 562–564. Publisher Full Text\n\nHammond JP, Broadley MR, White PJ, et al.: Shoot yield drives phosphorus use efficiency in Brassica oleracea and correlates with root architecture traits. J Exp Bot. 2009; 60(7): 1953–1968. PubMed Abstract \| Publisher Full Text\n\nGood AG, Shrawat AK, Muench DG: Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production? Trends Plant Sci. 2004; 9(12): 597–605. PubMed Abstract \| Publisher Full Text\n\nMasclaux-Daubresse C, Daniel-Vedele F, Dechorgnat J, et al.: Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Ann Bot. 2010; 105(7): 1141–1157. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBernard SM, Habash DZ: The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytol. 2009; 182(3): 608–620. PubMed Abstract \| Publisher Full Text\n\nMcAllister CH, Beatty PH, Good AG: Engineering nitrogen use efficient crop plants: the current status. Plant Biotechnol J. 2012; 10(9): 1011–1025. PubMed Abstract \| Publisher Full Text\n\nMonaco MK, Stein J, Naithani S, et al.: Gramene 2013: comparative plant genomics resources. Nucleic Acids Res. 2014; 42(Database issue): D1193–1199. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYan H, Jiang C, Li X, et al.: PIGD: a database for intronless genes in the Poaceae. BMC Genomics. 2014; 15(1): 832. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJin J, Zhang H, Kong L, et al.: PlantTFDB 3.0: a portal for the functional and evolutionary study of plant transcription factors. Nucleic Acids Res. 2014; 42(Database issue): D1182–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar AS, Hima Kumari P, Sundararajan VS, et al.: PSPDB: Plant Stress Protein Database. Plant Mol Biol Rep. 2014; 32(4): 940–942. Publisher Full Text\n\nWilkinson PA, Winfield MO, Barker GL: CerealsDB 2.0: an integrated resource for plant breeders and scientists. BMC Bioinformatics. 2012; 13: 219. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchreiber F, Colmsee C, Czauderna T, et al.: MetaCrop 2.0: managing and exploring information about crop plant metabolism. Nucleic Acids Res. 2012; 40(Database issue): D1173–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoodstein DM, Shu S, Howson R, et al.: Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 2012; 40(Database issue): D1178–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuvick J, Fu A, Muppirala U, et al.: PlantGDB: a resource for comparative plant genomics. Nucleic Acids Res. 2008; 36(Database issue): D959–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson C, Bowman L, Adai AT, et al.: CSRDB: a small RNA integrated database and browser resource for cereals. Nucleic Acids Res. 2007; 35(Database issue): D829–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKünne C, Lange M, Funke T, et al.: CR-EST: a resource for crop ESTs. Nucleic Acids Res. 2005; 33(Database issue): D619–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatthews DE, Carollo VL, Lazo GR, et al.: GrainGenes, the genome database for small-grain crops. Nucleic Acids Res. 2003; 31(1): 183–186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUniProt Consortium: Activities at the Universal Protein Resource (UniProt). Nucleic Acids Res. 2014; 42(Database issue): D191–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta PK: Competing endogenous RNA (ceRNA): a new class of RNA working as miRNA sponges. Curr Sci. 2014; 106(6): 823–830. Reference Source\n\nDai X, Zhao PX: psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res. 2011; 39(Web Server issue): W155–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar A, Pandeya A, Malik G, et al.: A Web-resource for Nutrient Use Efficiency related Genes, QTLs, and microRNA in important cereals and model plants [Data set]. Zenodo. 2018. Data Source" }	[ { "id": "34482", "date": "18 Jun 2018", "name": "Rajib Bandopadhyay", "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\nResolution of the figures are poor and better resolution figures must be supplemented.\n\nThere are some minor spelling mistakes in Page No. 3; like ‘levekls’ which should be written as ‘levels’.\n\nIn Figure 5, scientific name of the plant species should be written in italics.\n\nIn the manuscript, several grammatical mistakes are observed which can be amended in a way where the entire manuscript can be written in a same grammatical manner.\n\nOverall the NtUE web source is a good comprehensive catalogue on model cereal plant species, their growth and development and the manuscript has good scientific outcome.\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": "36463", "date": "02 Aug 2018", "name": "Vishal Acharya", "expertise": [ "Reviewer Expertise Plant bioinformatics", "genomics", "next generation sequencing", "machine learning" ], "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 well prepared the manuscript dealing with critical information about necessary for construction of database of nutrient use efficiency-related genes (NtUE) in cereals and model plants.\nThe authors suggested that this is first of kind to provide comprehensive information dealing with NtUE. The authors have compared also with the known database Gramene which is widely used and suggested the advantage in providing more information of model plants apart from cereals where Gramene only stand.\n\nThe overall conclusion is the paper is well researched and needs to be indexed.\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/7-673
https://f1000research.com/articles/7-243/v1	28 Feb 18	{ "type": "Opinion Article", "title": "Revised World Health Organization (WHO)’s causality assessment of adverse events following immunization—a critique", "authors": [ "Jacob Puliyel", "Pathik Naik", "Pathik Naik" ], "abstract": "The World Health Organisation (WHO) has recently revised how adverse events after immunization (AEFI) are classified. Only reactions that have previously been acknowledged in epidemiological studies to be caused by the vaccine, are classified as a vaccine-product–related-reaction. Deaths observed during post-marketing surveillance are not considered as “consistent with causal association with vaccine”, if there was no statistically significant increase in deaths recorded during the small Phase 3 trials that preceded it. Of course, vaccines that caused deaths in the control-trials stage would not be licensed. After licensure, deaths and all new serious adverse reactions are labelled as ‘coincidental deaths’ or ‘unclassifiable’, and the association with vaccine is not acknowledged. The resulting paradox is evident. The definition of causal association has also been changed. It is now used only if there is “no other factor intervening in the processes.” Therefore, if a child with an underlying congenital heart disease (other factor), develops fever and cardiac decompensation after vaccination, the cardiac failure would not be considered causally related to the vaccine. The Global Advisory Committee on Vaccine Safety has documented many deaths in children with pre-existing heart disease after they were administered the Pentavalent vaccine. The WHO now advises precautions when vaccinating such children and this has reduced the risk of death. Using the new definition of causal association, this relationship would not be acknowledged and lives would be put at risk. In view of the above, it is necessary that the AEFI manual be revaluated and revised urgently. AEFI reporting is said to be for vaccine safety. Child safety (safety of children) rather than vaccine safety (safety for vaccines) needs to be the emphasis.", "keywords": [ "Pentavalent vaccine", "quinvaxim", "pharmacovigilance", "Hill criteria", "macrophagicmyofasciitis", "periodic safety update reports", "Brighton classification", "adverse drug reactions", "sudden unexpected death", "TOKEN study" ], "content": "Introduction\n\nOne of the earliest countries to introduce the pentavalent vaccine (combined diphtheria, tetanus, pertussis, Hib, and hepatitis B) was Sri Lanka1. A pentavalent vaccine Quinvaxim (Crucell) was introduced in Sri Lanka on January 1, 2008. On the 29th of April that year the vaccine was withdrawn by the government following five deaths. A World Health Organization (WHO) team of experts investigated the adverse events following immunization (AEFI) and reported the deaths were “unlikely” to be related to vaccination. The full report was not widely available before it was presented to the High Court in Delhi, India2. From the full report it became clear that there was no alternate explanation for three deaths and thus, they should have been classified as “probable/likely” related to immunization, using the WHO Brighton criteria for classification of AEFI (see Box 1). The experts deleted the categories “probable” and “possible” from the AEFI Classification they used for assessment and then reported that the deaths were “unlikely” related to vaccination. The way the Brighton Classification was altered to enable this misleading classification of the deaths in Sri Lanka was reported in the Indian Journal of Medical Research and the British Medical Journal3,4. On May 4, 2013 the Ministry of Health of Vietnam suspended the use of Quinvaxim (Crucell) after it had caused 12 deaths5. The WHO experts investigated the Vietnam deaths. This time they reported, “Quinvaxem was pre-qualified by WHO…, no fatal adverse event following immunisation (AEFI) has ever been associated with this vaccine5.” This is the same brand of pentavalent vaccine that was used in Sri Lanka where WHO experts had previously documented AEFI deaths. It appears that after the Sri Lanka investigation and shortly preceding the Vietnam investigation, the methodology used for AEFI classification was revised. Using the revised AEFI causality assessment, AEFI reported from Sri Lanka could be classified as “Not a case of [AEFI].” From the WHO “Safety of Quinvaxem report,”5 it is apparent that previously documented deaths following immunization were removed from the records after this new categorization started.\n\nReference\n\nhttp://www.rho.org/files/rb3/AEFI_Causality_Assessment_WHO_2005.pdf\n\nReproduced with permission.\n\n\nHistorical background of causality assessment\n\nThe new mechanism that allows AEFI to be classified as “Not a case of [AEFI]” will be discussed.\n\nThe evolution of the logic of causality assessment is fascinating. Eminent philosophers, scientists, legal luminaries, and statisticians have grappled with the issue and a great deal has been written about it. It will be impossible to distil all of that for this write-up, except at the risk of oversimplification. As we are concerned primarily with assigning causality to alleged drug reactions, only some aspects of the debate are germane to this discussion.\n\nDefining cause and effect (X is the cause of Y) has not been easy. According to Hume6, the major features of causation are temporal precedence (X must precede Y), contiguity and regularity of the association of causes and their effects. Confounding, however, is possible by a third factor.\n\nIt is known that the consumption of ice cream is higher when there is a spike in the incidence of sunburns. One can conclude wrongly that eating ice cream can cause sunburns. The third factor in this case is hot weather conditions. Both eating ice cream and getting sun burnt are associated with sunny days. Hume avoided the confounding problem by stipulating that X can be considered as cause of Y only if X is sufficient for Y. That is however fallacious. Striking a match can light a fire only if there is oxygen. In itself, striking the match is not sufficient. The alternate position could be that X is cause of Y if, and only if, X is necessary for Y7. John Mackie suggested that in nature there could be multiple reasons (causes) for the same outcome8. Thus X may not be necessary for Y but at the same time, X may be sufficient for Y. A building may be set on fire by a spark from a short circuit in the electrical wiring (X) or as the result of an act of arson (Z). Thus neither (X) nor (Z) is necessary for Y, but both (X) and (Z) are sufficient causes for Y. The question then is whether Y would have occurred were it not for the factor X. This is known as the “but for” test. In jurisprudence, it has been acknowledged that where there are multiple causes working simultaneously the “but for” test is unworkable and the question of causality is whether the putative cause materially contributed to the result9. This has been argued in the case of Grahan Dickie V. Flexcon Glenrothes Limited [2009] ScotSC 143 (04 September 2009). Peter M. Willcock and James M. Lepp have discussed ‘Causation in medical negligence cases’ which elaborates on these issues.\n\nIn biology, there is further a probabilistic element to causation. If men of the same height and women of the same height were to have children, their children will not all be of the same height. For the same set of observed causal factors, there is probability distribution of possible heights7.\n\n\nAdverse drug reactions\n\nAdverse drug reactions (ADRs) can follow after the use of any drug. Careful evaluation is required to distinguish the events that are causally related to the drug from coincidental events. Causality assessment is crucial because the events could be iatrogenic and avoidable. Usually only a few react adversely to drugs on the market, whereas others are unharmed. The attribution of causality for such occasional happenings is particularly complex. Investigations of ADRs put causative association on a probability scale. The causality-assessment system developed by the World Health Organization Collaborating Centre for International Drug Monitoring is called the Uppsala WHO Centre (WHO UMC) Scale. This is widely used as it offers a simple methodology (see Box 2). In consonance with Hume’s postulates, the first step is to confirm temporal precedence and contiguity. The adverse event must appear after the suspected drug is administered and within a reasonable time-frame. Events where the time-to-drug-intake makes a relationship improbable are classified as “unlikely” to be related. Events within a reasonable time and for which there is no alternate explanation (which cannot be attributed to disease or other drugs) are classified as “probable/likely” related to the drug in question. Drug reaction is classified as “possible” where there is a reasonable time relationship, but for which there are also alternate explanations. In terms of John Mackie’s aphorism, the drug is considered sufficient but not necessary for the effect.\n\nReference The Uppsala Monitoring Center. The use of the WHO-UMC system for standardised case causality assessment. Reproduced with permission of Uppsala monitoring centre. Available at https://www.who-umc.org/media/2768/standardised-case-causality-assessment.pdf\n\nTo be classified as “very likely/certain” the reaction needs to be an objective and specific medical disorder or a recognized pharmacologic phenomenon, and there must be evidence of dose-related reaction or proof in terms of reappearance of symptoms on rechallenge. If death should occur as ADR, rechallenge is impossible. It is usually difficult to be certain about the causality of fatal ADR and the reaction is often classified as “probable/likely” or “possible.”\n\nThe difference between certain and probable/likely is simply the acceptable standard of proof. For “certainly,” a high-standard irrefutable proof is called for (falsification of the theory by a single irregular outcome). A single well-documented spontaneous rechallenge is strong evidence of regularity (even tough in just one patient). For “very likely,” the standard of proof is proof beyond reasonable doubt.\n\n“Balance of probability” is the level of proof needed to classify as “probable” or “possible” and this is the standard of proof, which is relevant to medicine and for pharmacovigilance. With this level of proof (prima facie true), the “Precautionary Principle” must be triggered. This is described later.\n\n\nAdverse events following immunization\n\nVaccines are drugs used as a preventive measure, given to entire cohorts of healthy persons. As they are administered in the absence of any disease, there is a very high expectation that it produce few adverse effects, and there is low tolerance for serious adverse events and deaths. Adverse events following immunization (AEFI) must be monitored more carefully than other drugs. A credible immunization safety evaluation and monitoring system is essential for the success of immunization programmes. The WHO developed the “Adverse Events Following Immunization (AEFI): Causality Assessment” otherwise known as the Brighton Classification. It is very similar to the WHO UMC causality categories for ADR. Until recently, this was the touch-stone used by WHO experts when AEFI were reported (see Box 1).\n\nOne measure of the sensitivity and responsiveness of this system is the alacrity with which the rotavirus vaccine RotaShield was withdrawn in 1999 after 12 cases of vaccine-induced intussusceptions were reported. About 1 in 2000 children younger than 2 months of age, develop intussusception from other causes. Based on the results of the investigations, the Centre for Disease Control (CDC) estimated that one or two additional cases of intussusception would be caused among each 10,000 infants vaccinated with the RotaShield vaccine. After about 100,000 infants were immunized, the vaccine was withdrawn10. In 2013, the methodology of AEFI evaluation was, however, revised.\n\n\nThe Council for International Organizations of Medical Sciences (CIOMS)/WHO: Report on vaccine pharmcovigilance\n\nIn October 2010 after a series of meetings, 40 experts (of whom 19 were industry representatives with possible conflicts of interest) helped rewrite the classification criteria for AEFIs. The document entitled “Definitions and Application of Terms for Vaccine Pharmacovigilance” is reported to “provide tools for higher excellence of signal detection and investigation of adverse events following immunization.”11\n\nOn page 170 of this 193-page document, under the heading “Notes for Guidelines,” it is stated in small print: “If there is adequate evidence that an event does not meet a case definition, such an event should be rejected and should be reported as “Not a case of [AEFI].” Such evidence is considered adequate, if an exclusion criteria is met, or an investigation reveals a negative finding of a necessary criterion (necessary condition) for diagnosis. Such an event should be rejected and classified as “Not a case of [AEFI].”11\n\nThe CIOMS/WHO “tool for excellence in signal detection” works by turning a blind eye to AEFI—classifying AEFI as “Not a case of [AEFI].” Not only is the causative association of AEFI to immunization denied, but it is made to appear the AEFI never occurred. Signal detection is no longer possible once AEFIs are removed from the system after being designated as “Not a case of [AEFI].” The story in the Introduction above where the WHO asserted in May 2013 that no fatal AEFI has ever been associated with Pentavalent vaccine5, suggests the Sri Lanka AEFI deaths2 are now reclassified as “Not a case of [AEFI]” using the CIOMS/WHO tool.\n\nAccording to the CIOMS/WHO report (page 11), a case definition can be adopted from the standard literature or by the reviewers themselves; not necessarily “an existing case definition.” The case definition helps draw on previous epidemiological research and facilitates further research to confirm a causal link. However, excluding causality in relation to an individual event cannot be dependent on that event conforming to a pre-existing case definition. It has been pointed out that the pejorative use of the term “rejected” (in the statement; ‘Such an event should be rejected and classified as “Not a case of [AEFI]”’), suggests a defensive posture. Reports of AEFIs are to be assessed for causality and classified, they are not to be “rejected”12.\n\n\nThe WHO revised AEFI manual\n\nIn March 2013, the revised WHO “User Manual for AEFI” was published with a new algorithm13. The manual acknowledges that it has adapted definitions and concepts from the CIOMS/WHO report. The new algorithm for AEFI is reproduced in Figure 1.\n\n\nRevised AEFI classification: New categories of causality\n\nOnly events that occur after vaccine administration are eligible for AEFI causality assessment. This first step is reminiscent of Hume’s dictum regarding precedence and contiguity. In the new scheme, causality is classified in four categories: “Consistent causal association to immunization,” “Indeterminate,” “Inconsistent causal association to immunization,” and “Unclassifiable.”\n\nThis is the highest level of causal association in this new classification. It is less definitive than “very likely/certain” in the old scheme. It does not call for irrefutable proof or even proof beyond reasonable doubt. Not even is the balance of probability assessed. In the new scheme, an adverse event can simultaneously be classified as “Consistent causal association with immunization” and “Inconsistent causal association with immunization.” On page 36 of the revised manual for AEFI13 is the example of acute flaccid paralysis in a child after oral polio vaccine, who had had a fever 1 month prior to onset of paralysis. The stool culture showed vaccine strain polio virus. It was classified as “Consistent causal association with immunization” as it is a known reaction and the paralysis happened within time window of increased risk. It was also classified as “Inconsistent causal association with immunization” because the fever, 1 month prior to paralysis had not been investigated completely. This ambiguity, which admits diametrically opposite conclusion simultaneously, is a hallmark of the new scheme.\n\nIt is suggested that before the question “Did the vaccine given to a particular individual cause the particular event reported?” (the question of ‘Did it?’) is answered, one has to answer the question “Can the given vaccine cause a particular adverse event?” (Can it?). The inference is that only if there is evidence at the population level that the vaccine can cause the adverse event, is the reaction classified as “Consistent with causal association with immunization.”\n\nThis inference is flawed on two grounds. On the one hand, it denies all new associations seen in Phase 4 trials. On the other, if it is a known adverse reaction, causal association is accepted even where the events could have happened by coincidence. Just because intussusceptions are acknowledged as an adverse event following rotavirus vaccination, it does not follow that all intussusceptions in the critical window of increased susceptibility are necessarily caused by it. The residual uncertainty at this highest level of causal association robs it of value in addressing the problem of AEFI caused by vaccines.\n\nAt the bottom of the new causality classification hierarchy is “Inconsistent causal association to immunization.” This group can include reactions for which there is no alternate explanation (and which would have been classified in the “Probable” category previously). They would fall in the group “Inconsistent causal association with vaccination” merely because causal association with immunization has not been documented in prior epidemiological studies. Into the same group are placed reactions that would have been considered “Unlikely” to be associated, and those that would have been classified as “Unrelated.” The use of the same category “Inconsistent causal association to immunization” for such a wide variety of clinical situations merely obfuscates the issues. In the revised scheme, this term is used to suggest that there is no relation between the AEFI and immunization. No matter how frequently the reaction categorized as “Inconsistent with causal association” occurs, it would not be investigated as a new signal of a causal association.\n\nClassification in the “Indeterminate” group is reserved for reactions that could have been caused by immunization, but for which causal association has not been documented previously. It is projected that information on AEFI that are classified as indeterminate will be pooled and analysed in order to understand if the AEFI represents a new signal of an unrecognized event. The scheme is however loaded such that literally no AEFI are categorized into this group. How this is accomplished is discussed later in this chapter.\n\nClinical events with insufficient information to permit assessment and identification of cause are put in the “Unclassifiable” group.\n\n\nRevised AEFI classification: The new algorithm\n\nJust as the final categories of causality association are vague, overlapping, and not clearly differentiated, the algorithm used to make a decision on causality13 does not appear to be logical or well thought through.\n\nThe algorithm is shown in Figure 1.\n\n\nCausality assessment algorithm\n\nFour sets of questions need to be answered in sequence:\n\n1. Is there strong evidence of other causes?\n\n2. Is there known causal association with the vaccine or vaccination and if so, whether the event was within the time window of increased risk?\n\n3. If there is no causal association known or if it is not within the time window of increased risk: Is there strong evidence against a causal association?\n\n4. If there is no such strong evidence against causal association, the next step is to look at other qualifying factors for classification.\n\na. Could it happen independently of vaccination (background rate)?\n\nb. Could the event be manifestation of another health condition?\n\nc. Did a comparable event occur after a previous dose of a similar vaccine?\n\nd. Was there exposure to a potential risk factor or toxin prior to the event?\n\ne. Was there acute illness prior to the event?\n\nf. Did the event occur in the past independently of vaccination?\n\ng. Was the patient taking any medication prior to vaccination?\n\nh. Is there biological plausibility?\n\nThe first step in the revised algorithm is to look for strong evidence for other causes. If there is an alternate explanation, the AEFI is classified as “Inconsistent with causal association to immunization.” John Mackie has noted that in nature there could be multiple reasons (causes) for the same outcome, and if two possible causes exist simultaneously either of them could be the causative factor8. It is to be noted that with the WHO UMC classification of ADR and the old WHO/Brighton Classification of AEFI, even if an alternate explanation is available, a causative association with drug or vaccine is still considered “Possible.” Moreover, the two possible causes could be working synergistically. An example of this is where genetic and other individual susceptibility factors make one susceptible to developing an AEFI14. In the new algorithm, if there is an alternate explanation for the AEFI, or another factor is involved15, causative association with vaccine is rejected13.\n\nThe COIMS/WHO Report on Pharmacovigilance is used at this level11. AEFI-specific case definitions for some reactions have been developed. In instances where specific case definitions and criteria are not available for a particular AEFI, it is permissible to improvise using case definitions adopted from “standard medical literature, or national guidelines or they may be adopted locally by the reviewers” (page 11 CIOMS/WHO report). AEFI that meet case definitions and which occur within the time window of increased risk are classified as “consistent causal association to immunization.”\n\nThe acceptable time window for each adverse event is different. For the macrophagic myofasciitis affected patients usually are middle-aged adults, presenting with diffuse arthromyalgias, chronic fatigue, and marked cognitive deficits, fatigue, or depression due to long-term persistence of aluminium hydroxide within macrophages at the site of previous immunization16. However, surveillance for Periodic Safety Update Reports (PSUR) do not extent for so long.\n\nTheoretically, reactions that are not known to have a causal association or those that are not in the time window of increased risk can move to Step 3. At this stage, an enquiry is made whether there is strong evidence against causal association. Proving of a negative is notoriously difficult as it is impossible to affirm that in every circumstance, an irregular outcome is impossible. The example provided in the manual relates to MMR and autism.\n\nIt is reported that the Global Advisory Committee on Vaccine Safety (GACVS) and Council for International Organizations of Medical Sciences (IOM committee) have concluded that no evidence exists of a causal association between MMR vaccine and autistic disorders. Such AEFI must be classified as “inconsistent with causal association to immunization” according to the new algorithm.\n\nAfter publication of this AEFI user’s manual, the conclusion about MMR and autism have become disputed again (see Box 3). This shifting evidence calls into question the usefulness of introducing this step in the algorithm of AEFI.\n\nIn 2004 the CDC published research demonstrating that there was no link between the vaccinated children’s risk of a subsequent diagnosis of autism and the age at which a child is vaccinated with MMRa. It has now been revealed through the testimony of one of the authors Dr. W. W. Thompson who turned whistle blower, that that the risk of autism among African American children vaccinated before the age of two years was 340% that of those vaccinated later. However this data was deliberately removed from the analysis to arrive at the CDC’s proclaimed conclusion. CNN published the story of the CDC whistle-blowerb, and Thomson has now been granted whistleblower immunity by the Obama administrationc.\n\nReferences:\n\na. DeStefano F, Bhasin TK, Thompson WW, Yeargin-Allsopp M, Boyle C. Age at first measles-mumps-rubella vaccination in children with autism and school-matched control subjects: a population-based study in metropolitan Atlanta. Pediatrics. 2004;113:259–66. doi: 10.1542/peds.113.2.259\n\nb. Goldschmidt D. Journal questions validity of autism and vaccine study [Internet]. CNN.com. 2014 Aug 28 [cited 2014 Sep 29]. Available from: http://edition.cnn.com/2014/08/27/health/irpt-cdc-autism-vaccine-study\n\nc. http://dailycaller.com/2015/02/03/obama-admin-grants-immunity-to-cdc-scientist-that-fudged-vaccine-report-whistleblower-plans-to-testify-before-congress/\n\nAssuming that no such “strong evidence against a causal association” exists, reactions that are not known to have a causal association with the vaccine, can go to Step 4. The question at this point is whether it is “classifiable”— meaning whether all the tests needed, have been performed to allow it to be classified under the CIOMS/WHO definitions. This is the second time these definitions are invoked during the AEFI evaluation.\n\nIf some investigations are not done or not available, the AEFI is labelled as “Unclassifiable” (or classified as “Inconsistent with causal association to immunization” like how flaccid paralysis following OPV was classified, because investigations during an illness 1 month prior to paralysis, were not available—(see Appendix 3, page 36 of the AEFI manual13 for this example).\n\nIf all the required investigations have been done and they did meet criteria, they would have been classified as “consistent causal association to immunization” at Step 2 and would not have come to Step 4.\n\nThe third possibility is that all the investigations had been done so it is classifiable but it did not meet case definitions. Bearing in mind the CIOMS/WHO definition, if there is adequate evidence that an event does not meet a case definition, such an event should be rejected and should be reported as “Not a case of [AEFI].” (See CIOMS/WHO Definitions and Application of Terms for Vaccine Pharmacovigilance, page 17011). It removes any chance that AEFI that has not been recognized as causatively associated with immunization in previous epidemiological studies will be included in the “Indeterminate” group and evaluated as a new signal.\n\nThe exercise does not end there. Other qualifying factors are also enquired into at Step 4. It is recommended that alternate explanations in terms of background rate, other health conditions, exposure to a potential risk factor or toxin, acute illness, and other medication are again enquired into. Many of these “other qualifying factors,” like prior illness and concurrent drug use would presumably have been eliminated at Step 1 when looking for evidence for other causes. This enquiry is repeated again at Step 4 quite unnecessarily. Box 4 illustrates how in spite of there being epidemiological evidence (the TOKEN Study) that pentavalent vaccine (as discussed in the introduction) can cause sudden unexpected death, the numerous deaths are not acknowledged as caused by the vaccine, and the WHO expert report denies that deaths were ever reported as AEFI. The causality assessment of 132 serious AEFI cases uploaded on the website of the Ministry of Health and Family Welfare in India illustrates the consequence of deploying this new classification. 54 of these babies died, whereas 78 survived. Not even one death was classified as vaccine-related, whereas the causality assessment found 50% of those who survived, had reactions to vaccination. Nearly all the deaths (96%) were simply classified as unclassifiable or coincidental17. Children admitted to hospital after vaccination with intractable convulsions, could be classified as having a vaccine-product related reaction, but if they died, the deaths would be classified as “coincidental deaths.”\n\nWith regard to AEFI a cluster of cases is defined as two or more cases of the same adverse event related in time or place or to the vaccine administereda. Pentavalent vaccine has caused numerous deaths in Asia but it is yet to be considered a new signalb–f.\n\nAfter the AEFI algorithm was revised, the deaths are now classified as 'Not a case of [AEFI]' on the grounds that deaths have not been reported as AEFIs in epidemiological studies involving the vaccine. However, the TOKEN study contradicts this assertiong.\n\nThe TOKEN study was done specifically to assess a possible causal relationship between vaccination and unexplained sudden unexpected death (SUD) of children between their 2nd and 24th month of life. vonKries had previously found a statistically significantly increased standardized mortality ratio (SMR) within two days after vaccination with one (Hexavac®) of the two licensed hexavalent vaccines and the TOKEN study was done to confirm or refute the associationg. The study was sponsored and supported by the Paul-Ehrlich-Institute (PEI) and the Federal Ministry of Health (Bundesministeriumfür Gesundheit).\n\nA self-controlled case series (SCCS) was examined to look for a temporal association of vaccination to SUD. Parents were invited to participate in the study if their child had died of SUD. 37.6% of the eligible parents participated. The researchers found that parents were twice as likely to participate if their child had died within one week of vaccination. They used an inverse probability weighted analysis to compensate for this bias. The authors note that this was helpful to overcome the selection bias for cases who died under 9 months, but even so, the results are still likely to overestimate the risk of SUD in older children.\n\nThe weighted SCCS analysis, relative risk of SUD after pentavalent vaccination(first and second year of life) looking at risk period 0-3 days after vaccination versus control period 4-28/183 days showed RR of 8.11 (p= 0.006, 95% CI=1.81-36.24; Table 41 in the TOKEN Report). The weighted SCCS analysis, relative risk of SUD after hexa- or pentavalent vaccination (1st and 2nd year of life) looking at risk period 0-3 days versus control period 4-28/183 days was RR.2.19 (p= 0.031, 95% CI=1.08-4.45; Table 36 in the TOKEN Report)\n\nIt is clear from the above that there is reasonable evidence in epidemiological studies that SUDS can occur as AEFI following use of the pentavalent vaccine and the deaths following the use of this vaccine should not be a priori classified as ‘Not a case of [AEFI]’.\n\nReferences\n\na. http://vaccine-safety-training.org/detection-and-reporting.html\n\nb. Lone Z, Puliyel J. Introducing pentavalent vaccine in the EPI in India: A counsel for caution. Indian J Med Res 2010; 132,: pp 1-3\n\nc. Puliyel J. AEFI and the pentavalent vaccine: looking for a composite picture. Indian J Med Ethics. 2013;10:142-6.\n\nd. No authors listed. Global Advisory Committee on VaccineSafety,12–13 June2013. Wkly Epidemiol Rec. 2013;88:301-12. Available at http://www.who.int/vaccine_safety/committee/reports/Jun_2013/en/ Accessed on 1/11/15\n\ne. Sreedhar S, Antony A, Poulose N. Study on the effectiveness and impact of pentavalent vaccination program in India and other south Asian countries. Hum Vaccin Immunother. 2014;10:2062-5.\n\nf. Martin Schlaud M, Poethko-Müller C, Kuhnert R, Hecker H Robert Koch Institute. Study on deaths in young children (2nd to 24th month of life) (TOKEN Study) http://www.rki.de/DE/Content/Gesundheitsmonitoring/Studien/Weitere_Studien/TOKEN_Studie/Studyreport.pdf?__blob=publicationFile\n\ng. vonKries R, Toschke AM, Strassburger K, Kundi M, Kalies H, Nennstiel U, Jorch G, Rosenbauer J, Giani G. Sudden and unexpected deaths after the administration of hexavalent vaccines (diphtheria, tetanus, pertussis, poliomyelitis, hepatitis B, Haemophiliusinfluenzae type b): is there a signal?. Eur J Pediatr. 2005;164:61-9.\n\n\nOther subtle changes in the definition of terms\n\nThe term causal association now means “a cause-and-effect relationship between causative factor and a disease with no factor intervening in the processes”. This is a major step backward for patient safety. The old scheme recognized, for example, that an elderly person with chronic cardiac failure might develop symptoms of cardiac decompensation after influenza vaccination due to a vaccine-caused elevation in temperature or stress from a local reaction at the site of vaccination. The vaccine is therefore considered to have contributed to cardiac failure in this specific situation18. Under the new scheme, this outcome would not be considered as causally related to the vaccine. The question of whether the death would have occurred at that time, had it not been provoked by immunization, would not be acknowledged. Without this recognition, many elderly persons may be exposed to this risk of death unnecessarily when using this vaccine. If the vaccination of an infant was reported to have been followed by sudden death but the child was malnourished or otherwise unwell it does not mean that causality assessment should conclude no cause and effect relationship between the vaccine and the death. There is no scope in this definition to consider interacting causalities12,14.\n\nAccording to Collet and colleagues, it is possible that some individuals experience greater immunogenic response to vaccines compared to the general population, and therefore, understanding genetically determined predispositions to developing AEFIs is important18. However, these considerations will not be accounted for in the new CIOMS/WHO causality assessment scheme. The contribution of vaccine in precipitating encephalopathy in patients who are susceptible on account of genetic factors will also not be considered14. Berkovic has used genetic analyses to identify de novo mutations in the sodium channel gene SCNIA in patients with alleged vaccine-induced encephalopathy15. Unwisely, in all these cases the contribution of the vaccine in precipitating the encephalopathy will be ignored.\n\nIt is a pity that after all these years, the authors should fall for the Hume fallacy that causality can be claimed only if X is sufficient in itself for Y. The fact that the immunization could have ‘materially contributed’ to the adverse events, is ignored.\n\nBox 5 describes how adverse events recorded in a randomized clinical trial (RCT) sent to the regulatory authority for vaccine approval and license are not made public. This goes against the European Court of Justice ruling that clinical study reports are made publically accessible.\n\nThe revised AEFI Categories further enabled vaccine manufacturers to classify AEFI not previously known to be associated with the vaccine in randomized clinical trials or other epidemiological studies as ‘Inconsistent with causal association to immunization.’\n\nVaccine trials and its reporting now seems designed not to report AEFI during the clinical trial.\n\nRotavirus trials in India\n\nRotaSheilds that was withdrawn as it caused 1 excess case of intussusception per 10,000 children vaccine10.\n\nHowever a new rotavirus vaccine Rotavac (Bharat Biotec ) was licenced in India after a trial in 3 centres where the vaccine was administered to a total of 4500 children (a sample size too small to show up a rare event that occurs 1 in 10,000). Even in spite of this small sample it appears intussusceptions were so common with this vaccinea in one of the centres (Vellore), it was significantly higher than controls. The trial doctors refused to provide this segregated data in spite of repeated requests for the same. The government promised to monitor safety in a post marketing surveillance. However the participants in this trial were not explained the risk seen in the RCT (as is mandatory for ethical clinical trials) and surveillance was for a limited window period of a few weeks after vaccination whereas the adverse events noticed in the RCT were outside that window period. In remote parts of this country where the vaccine is deployed, in the absence of paediatric surgeons and radiologists, deaths from intussusception are likely to be misclassified as deaths from dysentery.\n\nEven before this data of this post marketing surveillance is available, the WHO recently prequalified the vaccine to be used internationally.\n\nOther rotavirus vaccines that do not reduce incidence of diarrhoea or increase the incidence of diarrhoeac instead of decreasing it, have been publishedb.\n\nReferences\n\na. Bajaj J, Puliyel JM Intussusception risk with 116E rotavirus vaccine in Vellore, South India. Vaccine. 2016 Jan 20;34(4):403. doi: 10.1016/j.vaccine.2015.03.007. Epub 2015 Mar 21.\n\nb. Kulkarni PS, Desai S, Tewari T, et al. https://www.ncbi.nlm.nih.gov/pubmed/28967523\n\nc. Kaur J, Puliyel J. Heat-stable oral rotavirus vaccine. N Engl J Med 2017; 377:302\n\nOne of the qualifying factors considered at Step 4 is biological plausibility. The manual specifies that biological plausibility can only be invoked when laboratory findings or symptom or sign are similar or consistent with natural history and pathophysiology of the infection or antigen. Other biologically plausible explanations (demonstrating there is a mechanism and capacity to lead from the cause to the effect)7, do not qualify. One would have presumed that symptom or sign similar or consistent with natural history and pathophysiology of the infection or antigen would be “AEFI with known causal association with vaccine” and would have been picked up in Step 2 and would not reach Step 4.\n\nThe four approaches to ascertaining causality described by Brady include detection of neo-Humean regularity, examining the counterfactual, experimental manipulation and examining mechanisms and capacities7. The new AEFI recognizes only the experimental approach to the exclusion of other valid approaches and as a result can fail to detect causality in number of cases and result in harm.\n\n\nChronic fatigue syndrome and the HPV vaccine trial\n\nThe above discussion has assumed that adverse events that are reported in a statistically significant proportion of the population given the trial drug in the original prelicensure randomised control trials would be classified as adverse events known to be associated with the vaccine.\n\nSlate investigated of the randomised trials of human papillomavirus (HPV) vaccines and found that potential side effects were collected for only two weeks in the year long study. After the first 2 weeks, individual trial investigators decided on personal judgement whether to report medical problems as adverse events. Often they listed new problems as ‘new medical history. Myalgic encephalomyelitis otherwise known as chronic fatigue syndrome (CFS) characterized by long-term fatigue that limits a person’s ability to carry out ordinary daily activities. Participants in the trial reported to Slate that debilitating symptoms were not even registered as potential side effects.\n\nGiven that CFS was not recorded as an adverse event, it allowed the manufacturers to claim that CFS is not a ‘known adverse event with the vaccine’ and so to discount every case that was reported subsequently. Box 5 describes how trial data in a Rotavirus trial in India was concealed and the WHO approved the vaccine.\n\n\nOther problems with recording and reporting AEFI\n\nBox 6 describes how the Periodic Safety Update Reports (PSUR) 15 and 16 of Infanrix Hexa was opened to public scrutiny by an Italian court. Box 7 PSUR 19 was obtained under the Freedom of Information rules and shows how deaths reported in PSUR 16 were deleted from PSUR 19 when it was evident that the reported deaths exceeded the deaths expected by chance19. Box 8 describe the changes that prevent patients from holding manufacturers to account for adverse events caused by their products. Box 9 shows how AEFI data is no longer available easily. While on the one hand, the new classification discounts AEFI as ‘Not a case of [AEFI]’, safety data is being manipulated and made inaccessible.\n\nJustice Nicola Di Leo in Italy made public the ‘confidential’ 15th and 16th Periodic Safety Update Report (PSUR) on Infanrix hexa (GlaxoSmithKline Biological) and this is now available on the interneta\n\nPages 246-249 document an analysis of the number of ‘sudden deaths’ after receiving the vaccine, to examine if it exceeds the number of deaths one could expect from the natural background incidence of sudden death. The background incidence was calculated as 0.454/1000 in the first year and 0.062/1000 live births in the second year. No allowance is made for the notoriously poor AEFI reporting rate. The number of sudden deaths expected to occur by chance between day 1 and 20, is tabulated in Table 36 on page 24. The denominator used to examine deaths following vaccination is the number of doses of the vaccine distributed not the number of children vaccinated. This denominator would dilute any potential signal because many more vaccine doses are distributed than are actually administered!\n\nFurther, the number of doses actually administered may be appropriate for milder reactions that can recur with each dose, but it is not appropriate for deaths which can happen only once. Appendix 5A shows that 13 fatal cases were reported. There were more deaths after the first dose than after the second dose and third doses and the deaths after the second was more than after the third dose. This pattern is commonly seen with AEFIs that are causatively related. The appropriate denominator in all these cases is the number of babies vaccinated.\n\nThere were 42 deaths in the first three days after vaccination where there were only 16 deaths in the next 3 days. The fact makes it apparent that many of the deaths are related to the vaccination episode.\n\nPatient safety data should not be considered as trade secrets by any stretch of imagination. The practice of keeping safety reports confidential permits such data manipulation in a cozy relationship with the regulators, away from public scrutiny. Such practice ought to be reformed.\n\nReference\n\na http://autismoevaccini.files.wordpress.com/2012/12/vaccin-dc3a9cc3a8s.pdf Accessed 12/11/15\n\nGlaxoSmithKline (GSK), 19th confidential periodic safety update reportsa (PSUR 19 (deaths up to October 22, 2014)) on Infanrix hexa makes interesting reading. Infanrix hexa has all the components of the Pentavalent vaccine except that it has replaced the whole cell pertussis with an acellular pertussis component and in addition has injectable polio vaccine. The cumulative number of deaths after vaccination reported in the 19th report is less than that reported in the 16th PSUR. It can be seen that deaths in children older than 1 year was significantly higher than the deaths expected by coincidence, if the deaths deleted from the 16th PSUR were restored.\n\nIt appears that the EMA accepts PSUR reports filed by manufacturers, without reviewing them critically. Regulatory authorities internationally, rely on due diligence by the EMA in such circumstances. This may need to be reappraised.\n\nReference\n\na. http://ijme.in/wp-content/uploads/2017/09/infanrix-pusr.pdf\n\nHexavalent vaccine(DTaP-IPV-HepB/Hib) - Hexavac was withdrawn by the manufacturers without giving reasons after 5 cases of SIDS within 48 hours of receiving the vaccine were reported by Zinkaa. vonKries found that in the 2nd year of life, the standardized mortality rate (SMRs) for sudden unexplained deaths (SUD) cases within 1 day of vaccination with the vaccine were 31.3 (95% CI 3.8–113.1); and within 2 days after vaccination it was 23.5 (95% CI 4.8–68,6)b.\n\nSimilarly it will be noted that RotaSheild was voluntarily removed from the market after 12 cases of intussusceptions were reported. The background rate of intussusceptions at this age was 5 times the risk of intussusceptions from the vaccine. There was no biologically plausible explanation to link the intussusceptions to the immunization. Yet the vaccine was withdrawnc.\n\nThe manufacturers withdrew the vaccines voluntarily without indicating the reasons. Whether the prospect of product liability suits influenced manufacturer caution is not clear.\n\nTwo significant changes have taken place after 1980. The threat of vaccine manufacturers being held responsible for marketing a defective product has diminished greatly as a consequence of these changes.\n\n1. A no-fault compensatory mechanism has been put in many countries in the 1980s and 1990sd and this means that vaccine injured children need not provide clear evidence of negligence as cause of the harm, before they qualify for compensation. However, it also means that manufacturers do not have to admit to faults. The risk of product liability has now greatly decreased with no fault compensation being provided by Governments. As a result, manufacturers may be emboldened to be more reckless on vaccine safety issues.\n\n2. The second significant change was in 2013, when the methodology for assessment of AEFI was completely overhauled. It is no longer sufficient to show temporal association of the AEFI happening repeatedly. The flow diagram below depicts all conditions that need to be satisfied before an AEFI is labelled ‘Consistent causal association to immunization.’ This too could embolden manufacturers to be more reckless with regard adverse reactions.\n\nReferences\n\na. Zinka B, Rauch E, Buettner A, Ruëff F, Penning R. Unexplained cases of sudden infant death shortly after hexavalent vaccination.Vaccine. 2006;24:5779-80.\n\nb. vonKries R, Toschke AM, Strassburger K, Kundi M, Kalies H, Nennstiel U, Jorch G, Rosenbauer J, Giani G. Sudden and unexpecteddeathsafter the administration of hexavalentvaccines (diphtheria, tetanus, pertussis, poliomyelitis, hepatitis B, Haemophiliusinfluenzae type b): is there a signal?Eur J Pediatr. 2005;164:61-9.\n\nc. Center for Disease Control and Prevention.Rotavirus Vaccine (RotaShield) and Intussusception.http://www.cdc.gov/vaccines/vpd-vac/rotavirus/vac-rotashield-historical.htm\n\nd. Looker C, Kelly H. No-fault compensation following adverse events attributed to vaccination: a review of international programmes. Bulletin of the World Health Organization 2011;89:371-378. Available at http://www.who.int/bulletin/volumes/89/5/10-081901/en/ Accessed on 23/10/15\n\nIn 1986 President Ronal Reagan signed the National Childhood Vaccine Injury Act (NCVIA) (42 U.S.C. §§ 300aa-1 to 300aa-34 created a no-fault system to compensate vaccine related injuries. This made it difficult to sue vaccine manufactures. It also set up Vaccine Adverse Event Reporting System (VAERS) mandating the reporting of adverse events.\n\n1. Polio and Acute Flaccid Paralysis in India\n\nAs awareness of adverse events is increasing among the public it is becoming more difficult to access data on these adverse events. The National Polio surveillance provided monthly data on acute flaccid paralysis in India. An analysis of the data showed that in 2011, there an additional 47,500 children were newly paralysed in the year, over and above the standard 2/100,000 non-polio AFP that is generally accepted as the norm. The non-polio AFP rate during the year best correlates to the cumulative doses received in the previous three yearsa.\n\nThe analysis was repeated after 2 years when the number of doses administered to children under 5 was reduced and it showed the AFP rate had begun to declineb.\n\nHowever, the data is no longer provided on the National Polio Surveillance Project/WHO website.\n\n2. Data Analysis Prints on Vaccines\n\nMedicines and healthcare products regulatory agency of the government of UK (MHRA) provides easily accessible Drug Analysis Prints and interactive Drug Analysis Profiles (iDAPs)c from ‘Yellow Card’ notifications of adverse events but this is not provided for vaccines. One is required to request this MHRA Pharmacovigilance for this.\n\nReference\n\nVashisht N, Puliyel J. Polio programme: let us declare victory and move on. Indian J Med Ethics. 2012 Apr-Jun;9(2):114-7.\n\nVashisht N, Puliyel J, Sreenivas V. Trends in nonpolio acute flaccid paralysis incidence in India 2000 to 2013. Pediatrics. 2015 Feb;135 Suppl 1:S16-7. doi: 10.1542/peds.2014-3330DD. PMID: 26005734\n\n\nRevised AEFI classification and the precautionary principles\n\nIt is evident from the discussion earlier that the revised AEFI evaluation scheme produced by the CIOMS/WHO is designed to deny the possibility that any newly observed adverse event may be causally related to the immunization. The AEFI manual states “Allegations that vaccines/vaccination cause adverse events must be dealt with rapidly and effectively. Failure to do so can undermine confidence in a vaccine and ultimately have dramatic consequences for immunization coverage…”19,20.\n\nFigure 2 shows how all cases AEFI are classified as not causally related except those that are known adverse effects of vaccine.\n\nThe AEFI-denialism is a clear violation of the ‘precautionary principle’ (European Union law), which mandates that “when an activity raises threats of harm to the environment or human health, precautionary measures should be taken even if some cause and effect relationships are not fully established scientifically’. Society and Government is urged that until the full scientific evidence is available, where there is evidence of risk, it must take precautionary measures”. This new AEFI classification scheme that allows for an outright denial of any new causative association with vaccination could also fall foul of the Article 2 European Convention on Human Rights (Art 2 ECHR), which mandates governments to establish a framework of laws, precautions, and means of enforcement which will, to the greatest extend reasonably practicable, protect life.\n\nParadoxically the AEFI algorithm is said to be for vaccine safety. Perhaps we need a scheme for public safety rather than vaccine safety.\n\nThe story of Pentavalent vaccine was introduced at the beginning of this paper and is summarised in Box 10. It is primarily a vaccine used in developing countries where AEFI surveillance is poor, the press is less vigilant to report adverse events and where drug regulation is less strict. (The richer countries in West; Europe and the USA do not use the whole cell pertussis vaccine so this vaccine is not marketed in those countries). Isolated cases of unexplained deaths continued to be reported in the press. With the new AEFI classification, in the absence of ‘epidemiological evidence’ linking deaths to the vaccine, these deaths have been passed off as ‘coincidental’ SIDS deaths. Epidemiological evidence is now available linking the deaths to vaccine.\n\nThe story of Pentavalent vaccine\n\nIn 1949 the DTP vaccine was introducedaagainst diphtheria tetanus and pertussis. The first two were frequently fatal diseases. However, DPT was responsible for neurological adverse effects; seizures, encephalopathy, and hypotensive episodes (HHE)b. An acellular DTaP was developed and this has replaced DPT in the West.\n\nIn 1981 Hepatitis B was introduceda against this a viral infection that could lead to chronic liver disease and hepatocellular carcinoma (HCC) especially if acquired at birth. Vaccine uptake was poor in developing countries. One reason was that, although hepatitis B was common in the potentially large vaccine uptake countries like India, the incidence of HCC was very lowc. It is now thought that newborn babies in India may be protected in the early years (where the chance of becoming a chronic carrier is worst) by passive immunity from mother to babies. This may be lost once vaccine use becomes widespread and there could be a paradoxical increase in HCCd.\n\nIn 1987 Protein-conjugated Haemophilus influenza type b vaccine was introduced. The incidence of invasive disease in Asia is lowe perhaps due to cross protection other bacteria have cross-reactive antigens to the Hib capsular polysaccharidef. Hib vaccine uptake was poor in Asia.\n\nIt is said that the Pentavalent vaccine was introduced to improve the uptake of Hib and Hepatitis B, by combining new underused vaccines with a prior UIP vaccine like DTP as a way for the new vaccines to get a piggyback ride into the UIPg. The Pentavalent vaccine was used only in developing countries which had not switched to DTaP.\n\nPentavalent vaccine has been associated with deaths. In the investigation of deaths in Sri Lanka rather than reporting that the vaccine was probably related to the vaccine the WHO experts deleted the categories ‘probable’ and ‘possible’ from the Brighton classification. This ad-hoc improvisation was reported in medical journals. The AEFI classification was then formally revised so that reactions (deaths in this case) noticed for the first time in the Phase 4 trials (post marketing trials) could all be classified as “Inconsistent with causal association to immunization” and passed off as ‘coincidental SIDS deaths’.\n\nA new study involving 45 million infants given DPT vaccination and 25 million who received Pentavalent vaccine now provides epidemiological evidence that the odds of death after Pentavalent was doubled (OR 1.98 (95% CI 1.65 to 2.38)) compared to DPT. There were 122 additional deaths (95% CI: 101-145) within 72 hours, reported to the Government surveillance system, due to the switch from DPT to Pentavalent vaccine. A large number of these deaths could have been avoided had the AEFI manual not been revised and the AEFI were evaluated earlier. Protection against these disease could have been had even if the vaccines were administered separately. In fact combined DTP-Hepatitis B-Hib vaccine causes more there were more local reactions and it is less effective than when they were administered separatelyh.\n\nReferences\n\na. http://www.immunize.org/timeline/\n\nb. https://www.sciencedirect.com/science/article/pii/0264410X9190283C\n\nc. https://www.ncbi.nlm.nih.gov/pubmed/?term=Hepatitis+B+in+India%3A+Systematic+review+%26+report+of+the+_IMA+sub-committee+on+immunization\n\nd. https://www.ncbi.nlm.nih.gov/pubmed/29318526\n\ne. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2557750/\n\nf. https://www.ncbi.nlm.nih.gov/pubmed/?term=Natural+immunity+to+Haemophilus+influenza+b+in+infancy+in+Indian+children\n\ng. https://www.researchgate.net/publication/238069905_New_combination_vaccines_Backdoor_entry_into_India%27s_universal_immunization_programme\n\nh. https://www.ncbi.nlm.nih.gov/pubmed/19588375\n\nTo examine if deaths following Pentavalent vaccine were merely coincidental SIDS deaths, a study of 45 million infants given DPT vaccination and 25 million who received PV was undertaken. The study assumed that all the deaths associated (self-reported to the Government surveillance system with 72 hours of vaccination) with DPT could be coincidental SIDS deaths but any increase in the deaths rate after PV may be assumed to have been caused by PV. The odds of death after Pentavalent vaccine was doubled (OR 1.98 (95% CI 1.65 to 2.38)) compared to DPT. There were 4.7 additional deaths (95% CI: 3.5-5.9), per million vaccinated with Pentavalent vaccine instead of DPT (p<0.0001). By the time this evidence was put together 122 excess deaths (95% CI: 101-145) had been reported to the Government, due to the switch from DPT to Pentavalent vaccine. The contribution of the new AEFI classification in this delay in recognizing the problem is stark21.\n\n\nConclusions\n\nThat vaccines do more good than harm is taken as an article of faith – a dogma or tenet. The purpose of this exercise in AEFI-denialism is to prevent the undermining confidence in vaccines. However, the scheme does not seem to be working. Indeed, public scepticism seems to be increasing rather than diminishing with these efforts at reassurance that vaccines are safe22,23. Epidemics of vaccine preventable disease have resulted24.\n\nThe response in some States in the United States has been to make vaccination mandatory for admission to public schools. Personal and religious belief exemptions for vaccination are not be allowed in California, effective July 1, 2016. If the debates among US Republican Presidential aspirants are anything to go by, it is clear that there is a lack of widespread support for this measure. The Department of Health and Human Services Office for Civil Rights has now set up the Conscience and Religious Freedom Division to which individuals could complain if their conscience or religious freedom has been abridged. How these forces will interact is anyone’s guess, but the present scenario augur badly for public trust in vaccines and voluntary vaccination.", "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\nAcknowledgement of the help received from Lucija Tomljenovic for her inputs and suggestions during the drafting of this manuscript.\n\n\nReferences\n\nWHO: Global Vaccine Safety. Pentavalent vaccine in Asian countries. (accessed 20 February 2018). Reference Source\n\nWHO Experts Report: High Court of Delhi at New Delhi WP (C) 13698 of 2009 Dr KB Saxena and others (petitioners) versus Union of India and others (respondents). Submission of respondents dated 03/04/2010. [accessed 19.10.15].\n\nPuliyel J, Mathew JL, Priya R: Incomplete reporting of research in press releases: et tu, WHO? Indian J Med Res. 2010; 131: 588–9. PubMed Abstract\n\nSaxena KB, Banerji D, Qadeer I, et al.: “Antivaccine lobby” replies to the BMJ. BMJ. 2010; 341: c4001. PubMed Abstract \| Publisher Full Text\n\nWHO: Safety of Quinvaxem (DTwP-HepB-Hib) pentavalent vaccine. [accessed 15.10.15]. May 10, 2013. Reference Source\n\nHume DA: In: Selby-Bigge LA, Nidditch PH, editors. Treatise of Human Nature. Oxford: Clarndon; 1739. Reference Source\n\nBrady HE: Causality and explanation in social research. In The Oxford Handbook of Political Methodology. ed Janet M. Box-Steffensmeier, Henry E. Brady, and David Collier. Oxford University Press Oxford. Publisher Full Text\n\nMackie JL: Causes and conditions. American Philosophical Quarterly. 1965; 2: 245–64. Reference Source\n\nAthey v. Leonati (1996), 140 D.L.R. (4th) 235 at para. 32, [1996] 3 S.C.R. 458, [1997] 1 W.W.R. 97 quoted in David H, McCague WP, Yaniszewski PF The Advocates’ Quarterly. 2005; 30: 216–238. Reference Source\n\nCenter for Disease Control and Prevention: Rotavirus Vaccine (RotaShield) and Intussusception. Reference Source\n\nDefinitions and Application of Terms for Vaccine Pharmacovigilance CIOMS/WHO: Definitions and Application of Terms for Vaccine Pharmacovigilance Report of CIOMS/ WHO Working Group on Vaccine Pharmacovigilance. Reference Source\n\nLegge D, Puliyel J: Enhancing community confidence in vaccines safety. BMJ. 2017; 357: j2449. Reference Source\n\nWHO: The Causality assessment of an adverse event following immunization (AEFI): User manual for the revised WHO classification. WHO/HIS/EMP/QSS. March 2013. Reference Source\n\nSoriano A, Nesher G, Shoenfeld Y: Predicting post-vaccination autoimmunity: Who might be at risk? Pharmacol Res. 2015; 92: 18–22. PubMed Abstract \| Publisher Full Text\n\nBerkovic SF, Harkin L, McMahon JM, et al.: De-novo mutations of the sodium channel gene SCN1A in alleged vaccine encephalopathy: a retrospective study. Lancet Neurol. 2006; 5(6): 488–92. PubMed Abstract \| Publisher Full Text\n\nRigolet M, Aouizerate J, Couette M, et al.: Clinical features in patients with long-lasting macrophagic myofasciitis. Front Neurol. 2014; 5: 230. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPuliyel J, Phadke A: Deaths following pentavalent vaccine and the revised AEFI classification. Ind J Med Ethics. 2017; 2(4): 300–301. PubMed Abstract \| Publisher Full Text\n\nCollet JP, MacDonald N, Cashman N, et al.: Monitoring signals for vaccine safety: the assessment of individual adverse event reports by an expert advisory committee. Advisory Committee on Causality Assessment. Bull World Health Organ. 2000; 78(2): 178–85. PubMed Abstract \| Free Full Text\n\nPuliyel J, Sathyamala C: Infanrix hexa and sudden death: a review of the periodic safety update reports submitted to the European Medicines Agency. Indian J Med Ethics. 2018; 1(1): 43–47. PubMed Abstract \| Publisher Full Text\n\nGlobal Advisory Committee on Vaccine Safety,12–13 June 2013. Wkly Epidemiol Rec. 2013; 88: 301–12. PubMed Abstract\n\nPuliyel J, Kaur J, Puliyel A, et al.: Deaths reported after Pentavalent vaccine compared with death reported after diphtheria-tetanus-pertussis vaccine – an exploratory analysis. Med J DY Patil Univ. In print.\n\nMacDonald NE; ; SAGE Working Group on Vaccine Hesitancy: Vaccine hesitancy: Definition, scope and determinants. Vaccine. 33(34): 4161–4164. PubMed Abstract \| Publisher Full Text\n\nSci Dev Net: . Parental doubt drives drop in vaccination. http\n\nClemmons NS, Gastanaduy PA, Fiebelkorn AP, et al.: Measles - United States, January 4-April 2, 2015. MMWR Morb Mortal Wkly Rep. 2015; 64(14): 373–376. PubMed Abstract" }	[ { "id": "31301", "date": "14 Mar 2018", "name": "Tom Jefferson", "expertise": [ "Reviewer Expertise Tom Jefferson", "Clinical epidemiologist" ], "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 my views on this paper.\nThis is a very long and detailed examination of the philosophical, historical rationale and principles of causality assessment of possible vaccine harms, chiefly death. This is mixed with the narrative of the changes made by WHO to their own assessment rules.\nThe authors use several important examples to make their points.\nI regard this topic as extremely interesting and important and the authors should be congratulated for attempting to pull the main strands together, from David Hume to the Brighton Collaboration.\nDespite my interest I found the manuscript extremely heavy going with a difficult-to-follow thread. It soon became apparent that the authors think there has been something akin to an international conspiracy to bury the dead by changing the definitions of probable and likely causality. That may be so, but I could not find any convincing evidence in the paper.\nHere and there inaccuracies and typos add to the distractions. For example in box 10, DTP becomes DPT or the suggestions that Rotashield was withdrawn in 1999 as a consequence of the Brighton criteria. As far as I remember in 1999 we were setting up and had not produced the criteria or any other output yet.\nI would also check the data of Rotashield withdrawal from the market.\nWhat follows are a few suggestions to improve the manuscript (ms).\nFirst I would split the ms into 2 parts. One discussing the philosophical-historical basis for causality assessment perhaps as far as Brighton and the second one looking at the more recent changes.\nHere I have two further suggestions to offer.\nBradford Hill's criteria should be cited, even though they are not a perfect solution as Hill himself recognised. They should be cited because they have had an enormous influence on modern epidemiology (see Geoffrey Rose's variant for example) and because the formulation of some of them (temporality, strength, gradient) is very apt for vaccine exposure. Take temporality for example. The term AEFI which is so extensively cited concedes temporality when in fact temporality is only as good as the vaccination records. Often \"AEFI\" is used when we are not sure that exposure has taken place at all or that it preceded the clinical event/possible harm. So a balanced discussion of temporality (one of the absolute conditions for determining causality) must include absolute certainty or high probability that exposure preceded the event and that it had taken place at all.\n\nSecond I would offer the connection of probabilism and Fisherian theory with Hume's problem of induction. I see Fisher's work as the patch that allows us to go on with at least a partially clear conscience, as I do not think there is a solution to Hume's problem as nature is not (and never will be) universally uniform.\nI would tone down the plot theory rhetoric and would seek a written explanation from WHO for their actions. WHO do not have a good track record of answering researchers but the effort must be made and reported. Ditto for any other point which was unclear to the authors. I am not a great believer in plots, blunders fit the picture and my experience better, but the authors must try and get to the bottom of the rationale for the changes and, while at it, they might just want to ask WHO, CIOMS etc. to check the authors' facts and dates (but not their opinions of course).\nLast but not least please ask Brighton whether they were aware of WHO's actions (they must be) and what their views are.\nI hope these suggestions are useful to the authors.\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? 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": "3635", "date": "29 May 2018", "name": "Jacob Puliyel", "role": "Author Response", "response": "Reviewer 1 Referee Prof Tom JeffersonReviewer CommentAuthors’ responseThis is a very long and detailed examination of the philosophical, historical rationale and principles of causality assessment of possible vaccine harms, chiefly death. This is mixed with the narrative of the changes made by WHO to their own assessment rules.The authors use several important examples to make their points.I regard this topic as extremely interesting and important and the authors should be congratulated for attempting to pull the main strands together, from David Hume to the Brighton Collaboration.ThanksDespite my interest I found the manuscript extremely heavy going with a difficult-to-follow thread.It soon became apparent that the authors think there has been something akin to an international conspiracy to bury the dead by changing the definitions of probable and likely causality. That may be so, but I could not find any convincing evidence in the paper. ‘Conspiracy theory’ according to Barkun is a closed and unfalsifiable system and it is merely a matter of faith with no proof. We hope we have critiqued various provisions in the revised manual for AEFI and shown how it can lead to harm – how it can result in delays in the acknowledgment of the problems that can result from vaccines. We hope this is a critique of the revised manual ‘not unfalsifiable conspiracy theory’. According to the revised AEFI classification reactions must be ‘known to be associated with the vaccine’ before it is acknowledged as caused by the vaccine. We have shown how new signals can be (and are being) ignored on account of this proviso. We merely suggest there is potential for harm inherent in the revised system. Regarding the observation“international conspiracy to bury the dead by changing the definitions of probable and likely causality. That may be so, but I could not find any convincing evidence in the paper.”The WHO experts in Sri Lanka removed these categories (‘probable’ and ‘possible’) before they reported the deaths were ‘unlikely’ to be caused by vaccine. This is a verifiable fact. Had the categories not been removed, they would have had to report that 3 death (for with there was no alternate explanation) were ’probably’ related to vaccination. Chronologically at least, the new ‘Revised AEFI’ categories were developed after the Sri Lanka report was criticized in the BMJ etc. After the AEFI classification was revised, experts no longer have the mortification of having to report they have deleted ‘Probable’ and ‘possible’. The revised AEFI have eliminated the categories ‘probable/likely’ and ‘possible’. In fact the Sri Lanka experts were very keen to absolve the vaccine. They write they felt reluctant to even classify the deaths as ‘unlikely’ to be related to vaccine. I quote from the Sri Lanka report:“Unlikely: In defining this category, the panel took note of the fact that the WHO category ‘unlikely’ is often interpreted to mean that there is (conversely) some likelihood of a causal association between the adverse event and the vaccine(s) administered….” (The full report is uploaded here for easy accesshttp://www.jacob.puliyel.com/download.php?id=213) I have no doubt that the experts are motivated by a laudable desire to reduce vaccine hesitancy and the attendant risk of vaccine preventable disease. The reasoning for the revised AEFI categories must be similar to that of the experts of the Sri Lanka report. We have added a new paragraph explaining that we feel the motivation for the change was a laudable desire to reduce vaccine hesitancy. Here and there inaccuracies and typos add to the distractions. For example in box 10, DTP becomes DPT or the suggestions that Rotashield was withdrawn in 1999 as a consequence of the Brighton criteria. As far as I remember in 1999 we were setting up and had not produced the criteria or any other output yet.I would also check the data of Rotashield withdrawal from the market.The errors have been corrected in the revised manuscript. The point made by the reviewer about Rotashield is correct. It has been revised. As pointed out by the reviewer, RotaShield was marketed before Brighton was developed. The WHO/UMC system was in place at that time.Corrections have been made in the revised version.Bradford Hill's criteria should be cited, even though they are not a perfect solution as Hill himself recognised. They should be cited because they have had an enormous influence on modern epidemiology (see Geoffrey Rose's variant for example) and because the formulation of some of them (temporality, strength, gradient) is very apt for vaccine exposure. Take temporality for example. The term AEFI which is so extensively cited concedes temporality when in fact temporality is only as good as the vaccination records. Often \"AEFI\" is used when we are not sure that exposure has taken place at all or that it preceded the clinical event/possible harm. So a balanced discussion of temporality (one of the absolute conditions for determining causality) must include absolute certainty or high probability that exposure preceded the event and that it had taken place at all. Bradford Hill's criteria have been cited. Thanks.Second I would offer the connection of probabilism and Fisherian theory with Hume's problem of induction. I see Fisher's work as the patch that allows us to go on with at least a partially clear conscience, as I do not think there is a solution to Hume's problem as nature is not (and never will be) universally uniform.We did not take the reviewers suggestion to introduce Fisherian theory - to avoid making the write-up even more complicated. As the reviewer stated: the manuscript is already “extremely heavy going”I would tone down the plot theory rhetoric and would seek a written explanation from WHO for their actions. WHO do not have a good track record of answering researchers but the effort must be made and reported. Ditto for any other point which was unclear to the authors. I am not a great believer in plots, blunders fit the picture and my experience better, but the authors must try and get to the bottom of the rationale for the changes and, while at it, they might just want to ask WHO, CIOMS etc. to check the authors' facts and dates (but not their opinions of course).The revised manuscript states explicitly that the motivation for the changes is probably to reduce vaccine hesitancy. The WHO was contacted a year prior to publishing this article in the BMJ but there was no response (BMJ 2017;357:j2449, published 19 May 2017). They were again contacted after the first version of this manuscript was published on F1000Research, so they could respond in the comments section. There is no response so far (as of 12/4/18). Last but not least please ask Brighton whether they were aware of WHO's actions (they must be) and what their views are.Apparently the present Brighton team approves of these changes. They are now tasked with the responsibility to develop ‘case definition’ for ‘known AEFI’ (See CIOMS/WHO report)" } ] }, { "id": "31300", "date": "03 Apr 2018", "name": "Peter Aaby", "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 are to be complimented for having conducted this study. Proper handling of AEFIs is very important if we are to maintain trust between public health vaccinology and the community. However, I am missing the authors’ specific suggestions for how to improve the situation. As discussed below there are also details of presentation which could be improved.\nThere is a rather detailed description of the changes in the definition of causality in relation to the current concept of AEFI. However, I am missing some presentation of where is the AEFIs concept coming from historically and what is the underlying theoretical biological model of why AEFI might occur and how does that affect how AEFI are observed, reported and used. Furthermore, what are the regulators requirements? Apparently the dominant thinking is that vaccines only induce disease specific memory. Presumably genetic variability may in rare cases affect how this biological process takes place and this could cause specific AEFIs. What else are the causes of other AEFIs: co-incidental infections or chronic disease, co-administration of drugs or other vaccinations? Most of such events can presumably be rejected as not “caused” directly by the vaccine.\nHowever, the concept of vaccines may be changing. WHO experts have recognized that vaccines may have non-specific effects (NSEs) with consequences for child survival 1. Apparently, through epigenetic and metabolic changes, vaccines can reprogram the immune system and upregulate or downregulate both the innate and the adaptive immune system 2-5. If that is the case there is room for both beneficial and deleterious unexpected events following immunization (UEFI). Proper monitoring systems should also be able to detect beneficial UEFIs; for example, we have found that BCG reduces the risk of neonatal sepsis in low-birth weight children 6. On the other hand, DTP consistently increases female relative to male mortality, also in societies that have no sex-differential treatment 7. This is “unnatural” since there was no excess female mortality in the pre-vaccination era in West Africa 8. This being the case there should be room not only for the short-term AEFI as in the current system (14 days?) but also for much more protracted biological processes being classified as AEFI/UEFI. This would require new standards for how UEFI/AEFI are observed and registered.\nParallel with the description of the changes in the definition of AEFI, there is a series of examples where the authors apparently think there are real differences in mortality/safety issues between different vaccination groups. I have noted at least: Pentavalent vaccine and congenital heart disease; MMR and autism in African American children; Hexavac; Rotavac; HPV and chronic fatigue syndrome; Pentavalent vaccine vs DTP for SIDS. Sometimes these safety issues are mentioned in passing as examples in the discussion of the processes related to AEFI assessment. I found it sometimes unclear whether these example were presented in their own right as safety issues or whether they were only meant to illustrate problems in the assessment of AEFI, e.g. safety reports not forthcoming, etc. Sometimes the presentation was too short or unclear to be really convincing; for example, I had problems with the ROTAVAC story (box 5). It is unclear why it is said in Box 5: “Other rotavirus vaccines that do not reduce incidence of diarrhoea or increase the incidence of diarrhoea instead of decreasing it, have been published (b)”. The paper which is referenced apparently reported a 40% reduction in rota-diarrhoea. If the problem is that overall diarrhea was not reduced I think this can be present more clearly.\n\nI think the paper would be stronger/more convincing if the safety-issues that the authors believe have been documented as safety concerns were presented as safety-case stories in specific boxes; the effect of Pentavalent vaccine on SIDS is apparently such a concern. Then the text on the changes in the assessment of AEFI could refer to this or that AEFI problem which was illustrated in the safety-case stories. On the other hand if the story is about mismanagement of the assessment of AEFI, then the cases should be presented as such without implying a causal link between vaccination and AEFI; for example box 3 is an example of poor public communication but it has hardly been documented that MMR causes autism.\n\nAbstract:\nIt should not be assumed that ”Of course, vaccines that caused deaths in the control-trials stage would not be licensed.” RTS,S malaria vaccine was recently approved by EMA but the trial data indicate that RTS,S compared with control vaccines was associated with 2-fold higher mortality for girls 9,10. Neither the authors nor EMA apparently analysed the mortality data, overall or by sex.\n\nThe example with cardiac failure in children is not presented in the paper and should therefore not appear in the abstract unless it is fully described in the paper. The case might well warrant further presentation in the paper itself.\n\nIntroduction Being presented with the Sri Lanka and Vietnam cases in the first paragraphs, the reader is left wondering what was the implications of the WHO experts’ classifications. Was the pentavalent vaccine (Penta) reintroduced in the countries and how did that decision come about?\n\nCausality assessment In the long description of changes in the manual for AEFI assessment, it would be good to have an explanation of WHO’s own justification for these changes.\n\nSometimes the text appears to have been written some years back but have been maintained unchanged in in the current 2018-version. For example in Box 3 it is said that “Thomson has now been granted whistleblower status by the Obama administration”. By now this sentence should probably be: “Thomson was granted whistleblower status by the Obama administration”. Similar in the conclusion it is said that if the debates among Republican presidential aspirants “are anything to go by”. By now it can no longer be “are”.\n\nBox 10: this sentence has problems: “In fact combined DTP-Hepatitis B-Hib vaccine causes more there were more local reactions and it is less effective than when they were administered separately.”\n\nPage 10: Biological plausibility. There appears to be an increasing trend to dismiss “unexpected observations”/unpleasant observations with the argument that there is no “biological plausibility”. This was one of the arguments used by WHO experts to dismiss that high-titre measles vaccine (HTMV) could be associated with excess female mortality 11. There can obviously not be biological plausibility for a pattern just detected, that no one has ever thought about. The only relevant question is whether a pattern is repeatable – arguments about biological plausibility should not be allowed to dismiss observations of potential AEFIs. The excess female mortality was repeated in subsequent studies and WHO eventually withdrew the HTMV (1992).\n\nI found this sentence strange: “Slate investigated of the randomised trials of human papillomavirus (HPV) vaccines and found that potential side effects were collected for only two weeks in the year long study.”\n\nPage 13: “PV” has not been defined as the abbreviation for pentavalent vaccine.\n\nThe comparison of DTP and pentavalent vaccine is frightening. Please indicate whether it is SIDS death or all-cause deaths when it is said for example: “The odds of death after pentavalent vaccine was doubled”. Since it is your study I would have indicated that to the readers: “To examine if deaths following Pentavalent vaccine (PV) were merely coincidental SIDS deaths, we undertook a study of 45 million infants given DPT vaccination and 25 million who received PV”. Given the scary character of this report a bit more information on methods in data collection and analysis would be appropriate. Any hypothesis of why there would be a two-fold difference in SIDS (?) mortality? Did the patterns differ for boys and girls? We have found that DTP and Penta are both associated with much higher female-than-male all-cause mortality rates 7,12.\n\nConclusion I do not think the conclusion is really a conclusion to the content of the paper.\n\nHow do we proceed from here? How can we built a better system that finds even the AEFIs we do not want to see and had not expected – and at the same do not create mistrust in the vaccines (BCG, measles vaccines, OPV) which are associated with major reductions in child mortality in low-income countries. What time-frame should be used? AEFI should always be presented by sex. If there are sex-differential patterns of AEFI it might enhance the credibility of this patterns as a true AEFI since we have found sex-differential effects on mortality of most of common vaccines.\n\nBiological plausibility should not be used to dismiss any new and unexpected pattern. There is now evidence that vaccines may reprogram both the innate and the adaptive immune system epigenetically with effect on general susceptibility to non-targeted infections 2,5. Hence, the starting point should be that unlikely effects are likely because we have never examined the possibility.\n\nIt is standard practice in small safety study with deaths to dismiss them because we cannot see a connection. However, deaths following vaccinations should always be classified as potential-even-though-unlikely AEFIs. Otherwise we cannot accumulate the data and detect patterns we had not imagined. For example, when DTaP was tested in an RCT in Sweden there were 4 deaths among 2847 vaccinated children but none among 954 controls 13. Though the authors recognized that 4 deaths was too high and would have been significant if the whole Swedish population of eligible children had been used as controls, the study could find no link between the vaccine and the deaths. All properly conducted studies from low-income countries have found DTwP to be associated with increased child mortality 14-16.\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": "3637", "date": "29 May 2018", "name": "Jacob Puliyel", "role": "Author Response", "response": "Reviewer 2 Reviewer Professor Peter Aaby Reviewer Comment Authors’ response The authors are to be complimented for having conducted this study. Proper handling of AEFIs is very important if we are to maintain trust between public health vaccinology and the community. I am missing the authors’ specific suggestions for how to improve the situation. We thank the reviewer for his detailed review and this compliment. We attempt only to critique the revised AEFI classification. Before one makes an effort to improve it, there has to be an acknowledgement of the flaws in the present system. We make no claim to have developed an alternate system of classification. An appropriate body of experts will need to draft it, if there is a consensus on what is flawed with the present system. We have now introduced a new paragraph entitled: “Where do we go from here”. We have suggested that the WHO-UMC causality categories for drug reactions has stood the test of time (and the older Brighton system was adapted from it) may be used till a better system evolves. There is a rather detailed description of the changes in the definition of causality in relation to the current concept of AEFI. However, I am missing some presentation of where is the AEFIs concept coming from historically and what is the underlying theoretical biological model of why AEFI might occur and how does that affect how AEFI are observed, reported and used. Furthermore, what are the regulators requirements? Apparently the dominant thinking is that vaccines only induce disease specific memory. Presumably genetic variability may in rare cases affect how this biological process takes place and this could cause specific AEFIs. What else are the causes of other AEFIs: co-incidental infections or chronic disease, co-administration of drugs or other vaccinations? Most of such events can presumably be rejected as not “caused” directly by the vaccine. We have dealt very briefly with the historic and theoretical background – but within the 8000 word-limit, we could not deal with this in greater detail. Regarding: ‘AEFIs concept coming from historically’ The other reviewer Prof. Tom Jefferson (TJ) has suggested we introduce the contributions made by the AB Hill and we have made reference to that. We refer to genetic predisposition to AEFI in our article and also how underlying disease like congenital heart lesions can precipitate an AEFI. This paragraph has been added in the main body on the text. In the new version we make reference to non-specific effects of vaccine and we thank the reviewer for his suggestion. However, the concept of vaccines may be changing. WHO experts have recognized that vaccines may have non-specific effects (NSEs) with consequences for child survival 1. Apparently, through epigenetic and metabolic changes, vaccines can reprogram the immune system and upregulate or downregulate both the innate and the adaptive immune system 2-5. If that is the case there is room for both beneficial and deleterious unexpected events following immunization (UEFI). Proper monitoring systems should also be able to detect beneficial UEFIs; for example, we have found that BCG reduces the risk of neonatal sepsis in low-birth weight children 6. On the other hand, DTP consistently increases female relative to male mortality, also in societies that have no sex-differential treatment 7. This is “unnatural” since there was no excess female mortality in the pre-vaccination era in West Africa 8. This being the case there should be room not only for the short-term AEFI as in the current system (14 days?) but also for much more protracted biological processes being classified as AEFI/UEFI. This would require new standards for how UEFI/AEFI are observed and registered. We thank the reviewer for the references. In our new submission we refer briefly to these NSEs and the benefits and harms than can result. Parallel with the description of the changes in the definition of AEFI, there is a series of examples where the authors apparently think there are real differences in mortality/safety issues between different vaccination groups. I have noted at least: Pentavalent vaccine and congenital heart disease; MMR and autism in African American children; Hexavac; Rotavac; HPV and chronic fatigue syndrome; Pentavalent vaccine vs DTP for SIDS. Sometimes these safety issues are mentioned in passing as examples in the discussion of the processes related to AEFI assessment. I found it sometimes unclear whether these example were presented in their own right as safety issues or whether they were only meant to illustrate problems in the assessment of AEFI, e.g. safety reports not forthcoming, etc. Sometimes the presentation was too short or unclear to be really convincing; for example, I had problems with the ROTAVAC story (box 5). It is unclear why it is said in Box 5: “Other rotavirus vaccines that do not reduce incidence of diarrhoea or increase the incidence of diarrhoea instead of decreasing it, have been published (b)”. The paper which is referenced apparently reported a 40% reduction in rota-diarrhoea. If the problem is that overall diarrhea was not reduced I think this can be present more clearly. I think the paper would be stronger/more convincing if the safety-issues that the authors believe have been documented as safety concerns were presented as safety-case stories in specific boxes; the effect of Pentavalent vaccine on SIDS is apparently such a concern. Then the text on the changes in the assessment of AEFI could refer to this or that AEFI problem which was illustrated in the safety-case stories. On the other hand if the story is about mismanagement of the assessment of AEFI, then the cases should be presented as such without implying a causal link between vaccination and AEFI; for example box 3 is an example of poor public communication but it has hardly been documented that MMR causes autism. They are presented as potential safety problems that seem to get glossed over, by the Revised AEFI assessment methodology. (Please also see next point in row below). Regarding Rotavac the problem is that overall diarrhea is not decreased and this is clarified in the revised text Safety-case stories with Pentavac and Hexavac have been identified as such in the revised manuscript The MMR story Box 3 was about increased autism seen in African American boys vaccinated prior to age of 2 years (compared to those vaccinated after 2 years). Post-hoc, data of many African American children were excluded on the grounds that they did not possess a valid birth certificate –and reanalysis of this truncated data was published to suggest that MMR was not related autism in any group. This suggests a possible link between Autism and MMR (albeit in one specific ethnic and age group). In summary, all the examples noted by the reviewer in the article ( Pentavalent vaccine and congenital heart disease; MMR and autism in African American children; Hexavac; Rotavac; HPV and chronic fatigue syndrome; Pentavalent vaccine vs DTP for SIDS) are AEFI that are ‘probably’ related causatively with vaccination which are not acknowledged. In the case of MMR and Hexavac the new revised classification cannot be blamed as the data itself was falsified (Many African American boys excluded from MMR study and deaths deleted from PSUR 19). Abstract It should not be assumed that ”Of course, vaccines that caused deaths in the control-trials stage would not be licensed.” RTS,S malaria vaccine was recently approved by EMA but the trial data indicate that RTS,S compared with control vaccines was associated with 2-fold higher mortality for girls 9,10. Neither the authors nor EMA apparently analysed the mortality data, overall or by sex. The example with cardiac failure in children is not presented in the paper and should therefore not appear in the abstract unless it is fully described in the paper. The case might well warrant further presentation in the paper itself. This sentence has been corrected in abstract The case of heart failure in children is now included in the paper as suggested by the reviewer. Introduction Being presented with the Sri Lanka and Vietnam cases in the first paragraphs, the reader is left wondering what was the implications of the WHO experts’ classifications. Was the pentavalent vaccine (Penta) reintroduced in the countries and how did that decision come about? The vaccines were reintroduced after the ‘WHO experts’ report, and this is mentioned now in the revised manuscript. The inevitable follow-up question then is: Were there deaths after it was reintroduced? We know from the data from India that using the Revised AEFI manual, each death is certified as ‘inconsistent with causal association’ on the grounds that death has so far never been acknowledged as having occurred in epidemiological studies with the vaccine. This is explained in the paper with reference from literature. Causality assessment 1. In the long description of changes in the manual for AEFI assessment, it would be good to have an explanation of WHO’s own justification for these changes. 2. Sometimes the text appears to have been written some years back but have been maintained unchanged in the current 2018-version. For example in Box 3 it is said that “Thomson has now been granted whistleblower status by the Obama administration”. By now this sentence should probably be: “Thomson was granted whistleblower status by the Obama administration”. Similar in the conclusion it is said that if the debates among Republican presidential aspirants “are anything to go by”. By now it can no longer be “are”. Box 10: this sentence has problems: “In fact combined DTP-Hepatitis B-Hib vaccine causes more there were more local reactions and it is less effective than when they were administered separately.” 3. Page 10: Biological plausibility. There appears to be an increasing trend to dismiss “unexpected observations”/unpleasant observations with the argument that there is no “biological plausibility”. This was one of the arguments used by WHO experts to dismiss that high-titre measles vaccine (HTMV) could be associated with excess female mortality 11. There can obviously not be biological plausibility for a pattern just detected, that no one has ever thought about. The only relevant question is whether a pattern is repeatable – arguments about biological plausibility should not be allowed to dismiss observations of potential AEFIs. The excess female mortality was repeated in subsequent studies and WHO eventually withdrew the HTMV (1992). 4. I found this sentence strange: “Slate investigated of the randomised trials of human papillomavirus (HPV) vaccines and found that potential side effects were collected for only two weeks in the year long study.” 5. Page 13: “PV” has not been defined as the abbreviation for pentavalent vaccine. 6. The comparison of DTP and pentavalent vaccine is frightening. Please indicate whether it is SIDS death or all-cause deaths when it is said for example: “The odds of death after pentavalent vaccine was doubled”. Since it is your study I would have indicated that to the readers: “To examine if deaths following Pentavalent vaccine (PV) were merely coincidental SIDS deaths, we undertook a study of 45 million infants given DPT vaccination and 25 million who received PV”. Given the scary character of this report a bit more information on methods in data collection and analysis would be appropriate. Any hypothesis of why there would be a two-fold difference in SIDS (?) mortality? Did the patterns differ for boys and girls? We have found that DTP and Penta are both associated with much higher female-than-male all-cause mortality rates 7,12. 1. This has not been justified as far as we know. The rationale for revising the Brighton classification has also not been stated explicitly. Reviewer TJ suggested that WHO must be given an opportunity to defend the changes. David Legge and I had written to WHO, before we published the short critique in BMJ referenced in the paper (Reference 14). This is copied in the response to TJ. There was no response from WHO. This present paper was also sent to WHO after it appeared in F1000 to seek their comments. There has not been any response so far. 2. The reviewer is correct that the article has been written in parts and the tense needs to be corrected for consistency. This has now been done. 3. We thank the reviewer for this example that has been included in the text. 4. The Slate story: that was the point of the Slate report – that adverse events were not recorded properly. 5. Abbreviation PV has been removed 6. The DPT Pentavalent story is about deaths within 72 hours of vaccination. As babies taken for vaccination are usually not unwell, these must be considered as SIDS deaths in ‘well children’ and comparisons can only be made with the acceptable death rate for ‘well children’ and it must NOT be compared to the ‘all cause death rate’ which includes in the cohort well and unwell children. This is sometimes referred to as the ‘healthy vaccinee effect’ When we report “The odds of death after pentavalent vaccine was doubled” we DO NOT have to make any extra allowance for the ‘healthy vaccinee effect’ as both DPT and Pentavalent vaccine are given to healthy children. The deaths among children getting pentavalent vaccine was twice as high as those getting DPT There is no hypothesis for the deaths - except as explained in the Boatman case - that the use of multiple vaccines release more inflammatory cytokines (than when single vaccines given) which can act as neuro-modulators and can cause depression of the serotonergic 5-hydroxytryptophan (5-HT) system in the infant medulla and blunt the normal chemo-sensitive response to excess carbon dioxide and this can result in the death of vulnerable infants during sleep. The method of data collection is described in great detail in the reference (which would be too long to reproduce in this paper). It may suffice to say that both DPT and Pentavalent deaths were captured in the same ‘improved’ government surveillance system and the data has been made freely available on-line for rechecking by stake holders, and more studies by future researchers. The records did not specify sex of child. Conclusion I do not think the conclusion is really a conclusion to the content of the paper. How do we proceed from here? How can we built a better system that finds even the AEFIs we do not want to see and had not expected – and at the same do not create mistrust in the vaccines (BCG, measles vaccines, OPV) which are associated with major reductions in child mortality in low-income countries. What time-frame should be used? AEFI should always be presented by sex. If there are sex-differential patterns of AEFI it might enhance the credibility of this patterns as a true AEFI since we have found sex-differential effects on mortality of most of common vaccines. Biological plausibility should not be used to dismiss any new and unexpected pattern. There is now evidence that vaccines may reprogram both the innate and the adaptive immune system epigenetically with effect on general susceptibility to non-targeted infections 2,5. Hence, the starting point should be that unlikely effects are likely because we have never examined the possibility. It is standard practice in small safety study with deaths to dismiss them because we cannot see a connection. However, deaths following vaccinations should always be classified as potential-even-though-unlikely AEFIs. Otherwise we cannot accumulate the data and detect patterns we had not imagined. For example, when DTaP was tested in an RCT in Sweden there were 4 deaths among 2847 vaccinated children but none among 954 controls 13. Though the authors recognized that 4 deaths was too high and would have been significant if the whole Swedish population of eligible children had been used as controls, the study could find no link between the vaccine and the deaths. All properly conducted studies from low-income countries have found DTwP to be associated with increased child mortality 14-16. The conclusion has been revised as suggested We have included a paragraph on “Where do we go from here”." } ] } ]	1	https://f1000research.com/articles/7-243
https://f1000research.com/articles/7-672/v1	29 May 18	{ "type": "Research Article", "title": "Depression, quality of life and cortisol: a cross-sectional study of caregivers of patients with Alzheimer’s disease", "authors": [ "Emanuela Bernardi", "Katiuscia de Oliveira Francisco Gabriel", "Luana Bernardi", "Gláucia Renée Hilgemberg", "Elizama de Gregório", "Weber Cláudio Francisco Nunes da Silva", "Caryna Eurich Mazur", "Etiene Rabel Corso", "Juliana Maria Silva Valério", "Camila Diedrich", "Juliana Sartori Bonini", "Emanuela Bernardi", "Katiuscia de Oliveira Francisco Gabriel", "Luana Bernardi", "Gláucia Renée Hilgemberg", "Elizama de Gregório", "Weber Cláudio Francisco Nunes da Silva", "Caryna Eurich Mazur", "Etiene Rabel Corso", "Juliana Maria Silva Valério", "Camila Diedrich" ], "abstract": "Background: Stress can impact human health in multiple ways. Among the related mechanisms are the hormonal systems of the hypothalamic–pituitary–adrenal axis, which produces cortisol. Current research aims to evaluate the relationship between the daily variation of salivary cortisol dosages and the level of stress in caregivers of patients with Alzheimer's disease (AD). Methods: A sociodemographic questionnaire was applied to 25 caregivers, as well as the 36 Item Short-Form Health Survey and Beck’s Depression Inventory. In the 25 patients of the caregivers, the Instrumental Activities of Daily Living of the patient and Clinical Dementia Rating were assessed. Saliva samples were collected to assess the cortisol level of the caregivers three times over one day for each caregiver, (morning, afternoon and evening) to investigate the correlation of the aforementioned questionnaires with the age and degree of kinship among caregivers of elderly patients to investigate the correlation with the results of the previously described tests, and the age and degree of relatedness of caregivers and elderly patients. Results: There was a significant positive correlation between daily cortisol levels and increasing caregiver age. However, the daily dosage of salivary cortisol was not significantly associated with the stress level of the caregivers of patients with AD, suggesting that this is not a good neuroendocrine marker of response to mood disorders. This fact can be related to intrinsic and extrinsic factors to the caregiver. Conclusions: Compared with previous studies that correlate cortisol and stress in humans, our findings suggest that the stress mechanism may be more complex and depend on more factors than the levels of this hormone. Thus, further work is required to delineate possible cortisol modulators, as well as the type of stress that target this population and their ability to adapt and face adversity in their work.", "keywords": [ "salivary cortisol", "caregivers", "cognitive deficits" ], "content": "Introduction\n\nPopulation aging brings with it an increase in the incidence and prevalence of dementias, such as Alzheimer's disease (AD)1. AD is characterized by a progressive decline in cognitive and functional abilities, requiring an increasing need for clinical care of patients2,3. Studies have shown that these caregivers are subject to chronic diseases caused by their intense work routine with patients4–6.\n\nResearch indicates that stress can lead to a number of changes in the individual's brain7,8. Deficits in the domains of attention, working memory and executive function, mediated by the prefrontal cortex7 have already been verified in caregivers of patients with dementia9,10. In addition, the impairment of declarative memory dependent on the hippocampus region may also result from the chronic physical and emotional overload suffered by these caregivers11,12.\n\nThe main reason for the cognitive impairment of chronically stressed individuals is hypothalamic–pituitary–adrenal (HPA) axis dysfunction coupled to changes in glucocorticoid levels, especially cortisol, and changes in lower affinity receptors of these hormones. Hypercortisolemia, besides being neurotoxic when prolonged13, may induce changes in the morphology and physiology of structures related to cognitive functions in the organism14.\n\nAnother condition related to prolonged periods of stress, excessive release of glucocorticoids and hyperactivity of the HPA axis is in hypocortisolemia, induced in these cases by failures in the ability of self-adjustment of allostatic systems15. Two theories justify the etiology of hypocortisolemia; the first correlates hypocortisolemia with the increase of the negative feedback sensitivity of the HPA axis16 and the second relates to adrenal insufficiency, caused by the chronicity of the response. Thus, the individual initially develops hypercortisolemia; however, the HPA axis collapses or fatigues and leading to hypocortisolemia15.\n\nAllostatic biological mechanisms usually develop in the organism in an individual way based on the experiences lived by the individual, especially in the form of adaptive responses to stress. These are usually coordinated and involve different functional systems, particularly the nervous, endocrine and immune systems. Regarding the functioning of the HPA axis, research shows that as adults experience life events associated with stressful problems, innumerable biological triggers are developed that are associated with the incidence of psychopathologies17.\n\nThere is a growing body of evidence linking hormones besides cortisol as being responsible for stress responses, including progesterone and dehydroepiandrosterone (DHEA)18, and brain-derived neurotrophic factor (BDNF)19–24. Improved understanding of stressors, their mechanisms and co-influences are essential for a better understanding of the physiology of stress and of psychological disorders that are implicated in this physiological condition. Thus, the objective of this study was to evaluate the relationship between the daily variation of salivary cortisol dosages and the level of depression in caregivers of patients with AD.\n\n\nMethods\n\nThe study population consisted of caregivers of elderly patients with AD (n=25) and patients admitted to the Association for Research and Assistance to Patients with Alzheimer's (AEPAPA) (n=25) in the city of Guarapuava (PR). In order to determine the size of the sample, we considered all patients that fit the inclusion criteria of the study and are serviced by AEPAPA. In this study, the age cut-off used to classify patients as elderly was based on the criteria proposed by the World Health Organization (WHO), which defines individuals aged 60 years or older from developing countries as elderly. In order to identify and select the caregivers, AEPAPA was asked for an official list, containing identification, address and telephone number of the registered elderly. Subsequently, the cadastral data was confirmed with the Social Worker, with the intention of eliminating the names of the deceased or who had changed their residence or changed their city. A telephone appointment was initially made by the social worker of the AEPAPA. In case of refusal or absence (three attempts were made to contact each domicile), the subject was excluded. On the day of the visit, the professional, together with a nurse from the AEPAPA, made the verbal invitation to participate in the research, by means of a previous explanation of all the stages of the study and, later completing the Informed Consent Form (TCLE) by the participants. In the case of illiterate caregivers, the informed consent form was signed by a family member responsible after reading the terms to the caregiver. AEPAPA is a Civil Society with a care purpose, responsible for providing home care to the elderly, with Public Utility, according to Law No. 2157/2013, contained in the Official Gazette of the Municipality from August 24 to 30, 2013. The ethical precepts of voluntary, enlightened and consensual participation of each participant were respected through a Free and Informed Consent Term – informed consent form signed by the participant (caregiver and patient). The project was approved by the Ethics Committee on Human Research of the Health Sciences Sector, the State University of West Paraná Center (896 296/2014).\n\nThis descriptive cross-sectional study began with the collection of data and salivary material from caregivers in the home of the elderly with AD, by a team composed of a nurse, a psychologist, a social worker, a pharmacist and a nutritionist from AEPAPA in the period from December 2014 to December 2015. The study was quantitative and used formal instruments to collect the data as questionnaires, scales and tests based on the work of 25. Initially, the caregivers who agreed to participate were asked to refrain from eating, drinking caffeine, brushing their teeth and vigorous exercise for 2 h before the collection of cortisol.\n\nParticipants provided the first saliva sample in the morning, between 08:00 and 09:00 hours, after which the questionnaires were applied to the caregiver and the elderly patient. Initially, the sociodemographic characterization of the caregivers was based on the following aspects: relationship to the elderly patient, age, race, marital status, number of children, schooling, income, type of housing, hygiene conditions and basic sanitation in the residence where they performed their care work. The remaining tests included information such as the analysis of the caregiver’s quality of life through the 36-Item Short Form Survey (SF-36) and the investigation of affective and somatic cognitive behavioral manifestations in the face of a possible picture of depression presented by the caregiver with the application of the Beck Depression Inventory (BDI), as well as the evaluation of the Instrumental Activities of Daily Living of the patient (IADL) with the help of the Lawton Scale, which focuses on assessing the elderly person's ability to maintain an independent life that interferes with the degree of caregiver activity, and classification of the stage of Alzheimer's disease using the Clinical Dementia Rating (CDR)26–29.\n\nThe other saliva samples were collected in the afternoon, between 16:00 and 17:00 hours, and at night, between 22:00 and 23:00 hours, taking into account the circadian fluctuations of cortisol. After collection in a Salivette tube (Salivette®, Sarstedt, Alemanha), since the biochemical dosage was performed on the saliva, the samples were immediately stored in a test tube (typically Ultra-High Performance 15 ml centrifuge tubes; VWR, Radnor, PA). At the end of the sample collection, the samples were sent to the Master Laboratory, a private clinical laboratory, in Guarapuava, PR, with no charge on the patient.\n\nIn the laboratory, the samples were refrigerated in a freezer (-4°C) for 24 hours until processing. Extraction of Salivette cotton saliva was performed by centrifugation (10 min at 3000 rpm) being the cortisol dosage in the saliva was performed by electrochemiluminescence immunoassay according to the instructions of the manufacturer (ECLIA; catalog number 11875116160; Roche Diagnosis GmbH, Mannheim, Germany).\n\nThe SF-36 questionnaire, applied to caregivers in the study, has the advantage of being easy to understand and requiring a short application time; therefore, it is well suited to assessing the associations of several types of diseases with the quality of life of those involved. SF-36 consists of 36 general questions grouped into eight domains: functional capacity (10 items), vitality4, physical4, pain2, general health5, social2, emotional3 and mental health5. Each of these questions is answered with a value between 0 and 100, where 0 is the worst state of health and 100 is the perfect state of health26.\n\nThe BDI, applied to caregivers in the study, is an instrument structured in 21 descriptions of affective and somatic cognitive symptoms of depression, with answers to each question ranging from 0 to 3 points dealing with the absence of depressive symptoms until the presence of symptoms. Originally created by Aaron Temkin Beck30, satisfactory evidence of its trustworthiness and validity was shown by a study by Cunha, 200131. To get a total score to be evaluated, the points of each item are added, giving a total score whose maximum does not exceed 63 points. According to Caixeta, scores from 0 to 9 points indicate an absence of depression or minimal depressive symptoms; from 10 to 18 points indicate mild to moderate depression, from 19 to 29 points indicate moderate to severe depression, and 30 to 63 points indicate severe depression28.\n\nThe evaluation of the implementation capacity of IADL was performed by applying the Lawton Scale to patients with AD in the study. For each task, there are three possible answers, with scores ranging from 1 to 3 (1, dependence; 2, ability to perform the task with help; and 3, independence). The final score is reached by the sum of points of the eight domains and ranges from 8 to 24; the higher the score, the more independent the individual27,32.\n\nThe AD CDR scale, applied to elderly patients in the study, was developed in 1979 at University of Washington's, St. Louis, Missouri, through the project\" Memory and Aging\"33, to graduate dementia and classify patients according to the disease stage, evaluating the presence or absence of cognitive impairment on a five-point scale (0, 0.5, 1, 2, 3). The six domains analyzed by the test were: memory, orientation, judgment and problem solving, community function, home and hobby function, and personal care. A general CDR score was then performed using the individual ratings in the 6 areas, according to the standard scoring rules34; so that a CDR score of 0 indicates no dementia, while CDR scores of 0.5 1, 2 and 3 indicate very mild, mild, moderate and severe dementia, respectively. In addition, individual domain scores can be summed, totaling a score ranging from 0 (0×6: when there is no impairment in any domain) to 18 (3×6: when there is maximum commitment in all domains)29.\n\nTo describe the results, descriptive tests were carried out with measures of central tendency and frequencies. To verify normality of the sample, the Shapiro–Wilk test was calculated. Pearson’s and Spearman’s correlation analyses were applied to parametric and non-parametric data, respectively. Also, for the association between CDR and cortisol values, the Kruskal–Wallis test was applied. The level of significance was set at 5% (P<0.05). The analyses were performed using the Statistical Package for the Social Sciences software version 22.0.\n\n\nResults\n\nOf the total sample, 88.0% (n=22) of the caregivers were female, 68.0% (n=17) of whom were Caucasian. Regarding marital status, 56.0% of participants (n=14) were married and 84.0% (n=21) had up to 4 children, while 16.0% (n=4) reported having 5 to 8 children. Regarding the level of education, 8.0% of caregivers (n=2) were illiterate, 44.0% (n=11) did not complete elementary school and only 4.0% (n=1) had completed higher education. Regarding the type of housing, 92.0% (n=23) resided in brick houses, while 8% (n=2) reported living in wooden houses. 76.0% (n=19) rated their hygiene conditions as good, compared to 24% (n=6) who reported very good hygiene conditions; all caregivers had water, electricity and sewage in their homes. As to income, 88.0% (n=22) of caregivers reported monthly income less than two minimum wages, equivalent to US $583.00. By contrast, only 12% (n=3) reported an income of up to three minimum wages.\n\nThe distribution of participants in terms of their relationship to the patients was predominantly close to the patients, with 44.0% (n=11) and 24.0% (n=6) of caregivers self-described wives or husbands, respectively. Caregivers were family members in 80.0% (n=20) of cases; among these, 24.0% (n=6) of caregivers self-declared as wives or husbands, 8.0% (n=2) as grandchildren and 4.0% (n=1) nephews. The remaining 20.0% (n=5) were unrelated caregivers.\n\nWith respect to depression affecting caregivers, values obtained from the BDI resulted in a mean score of 15.68 (SD=10.05), indicating mild to moderate depression in 36.0% (n=9) of the caregivers, while 20.0% (n=5) of caregivers were classified as having moderate to severe depression, 12.0% (n=3) as having severe depression and 32.0% (n=8) as not having depression.\n\nSF-36 variables showed close variations between functional capacity and social aspects among caregivers, with a mean score of 74.2 (SD=28.7) for the functional capacity and 64.4 (SD=28.1) for the social aspect. Regarding functional capacity, the variable with the lowest mean score was the physical aspect (mean=50, SD=46.8), compared to the aspects of pain (mean=53, SD=22.9), general health status (mean=52.7, SD=15.6) and vitality (mean=53.2, SD=19). As for the social aspects, the emotional variable (mean=40, SD=48.1) had the lowest score compared with mental health (mean=54.2, SD=21.7).\n\nIn the evaluation of IADL, the results showed that 70.83% (n=17) of patients with AD were not able to maintain an independent life, and 20.83% (n = 5) were unable to perform any of the nine corresponding functions of the scale. In total, 50.0% (n=12) performed some activity with partial help, and 29.17% (n=7) were able to perform the activities without help. One patient did not participate in the questionnaire.\n\nRegarding the stage of the disease 16.0% (n=4) were in the mild stage of AD (CDR 1), 52.0% (n=13) in the moderate stage (CDR 2) and 32.0% (n=8) in the severe stage (CDR 3).\n\nFor the salivary cortisol test, 3 samples were collected from each patient, accounting for a total of 75 samples. With regard to the salivary cortisol dosage in the morning, the median value was 0.50 μg/dl, with a minimum of 0.33 μg/dl and maximum of 1.27 μg/dl (reference value, approximately 0.69 μg/dl). Salivary cortisol measured in the afternoon resulted in a median value of 0.32 μg/dl, with a minimum of 0.09 μg/dl and a maximum of 0.71 μg/dl (reference value, approximately 0.43 μg/dl). For the tests performed in the evening, the median value was 0.68 μg/dl, with a minimum of 0.32 μg/dl and a maximum of 1.26 μg/dl (reference value, approximately 0.35 μg / dl). The cortisol values were analyzed according to the values cited by Aardal and Holm, 199535.\n\nAs analyzed in the paragraph above, the percentage of caregivers who presented cortisol at normal concentration was considerably higher than the percentage of caregivers who exhibited cortisol at an altered concentration, in the collection periods (morning, afternoon and night). In the morning, 80.0% (n = 20) of the caregivers did not present alterations in cortisol dosages, according to the reference values used35, whereas in the afternoon the percentage dropped to 72.0% (n = 18) and 60.0% (n = 15) at night, according to the circadian cycle of cortisol, evidenced in healthy individuals.\n\nOf the variables analyzed by the study, only the age of the caregivers was significantly correlated with the dose of salivary cortisol collected in the afternoon (r=0.470; P=0.018), as well as when it was correlated with daily cortisol variation, corresponding to mean of the cortisol dosage in the three periods (r=0.403; P=0.046) (Table 1).\n\nPearson’s correlation coefficient. Spearman’s rank correlation. *p<0.05 vs. age. IADL, Instrumental Activities of Daily Living. BDI, Beck Depression Inventory. SF-36, 36-Item Short-Form Health Survey; CDR, clinical disease rating.\n\nFigure 1, Figure 2 and Figure 3 show a positive correlation but no significant variation between nocturnal salivary cortisol (μg/dl) and the scores from AIVD, IDB and SF-36. Figure 4 shows a positive and significant correlation (r=0.403; P=0.046) between the daily variation of cortisol dosage (μg/dl) and the age of caregivers.\n\nAll correlations with significant p values were considered weak, owing to the sample size. However, it is possible to observe that cortisol values in the morning were correlated with the degree of kinship (r=0.289; p<0.05). The correlation was weak but inverse between the degree of kinship and the daily variation of cortisol (R²=-0.311; p<0.05).\n\nTable 2 shows the relationship between the CDR scores of the patients with AD and the corresponding mean salivary cortisol doses (mg/dl) of their caregivers. Mean values of salivary cortisol of the caregivers, measured in the morning and afternoon periods, remained below the cortisol values considered normal for these periods, which are approximately 0.69 μg/dl and 0.43 μg/dl, respectively, for the three groups of investigated CDRs. This diverged from the mean cortisol scored at night, which remained above the reference value considered normal for this period, approximately 0.35 μg / dl, for the three analyzed groups.\n\nData are expressed as the mean ± SD. Calculated using the Kruskal–Wallis test.\n\nIn a horizontal analysis of the table, the mean cortisol levels in the specific periods evaluated (morning, afternoon and evening) and the mean daily cortisol variation did not change significantly (P>0.05) when associated with the CDR groups (Table 2).\n\n\nDiscussion\n\nOur study showed that daily salivary cortisol levels did not differ significantly when associated with the level of depression in caregivers of patients with AD, suggesting that this is not a good marker of neuroendocrine response to mood disorders. It is understood that this may be related to intrinsic and extrinsic factors to the caregiver.\n\nWe found that the emotional caregiver burden, as measured by the BDI and SF-36 tests, was also not reflected in the levels of cortisol daily. Maria and Jeckel concluded that caregivers were more anxious, depressed and stressed than healthy, non-caregiving controls, although there was no increase in daily levels of salivary cortisol. On the other hand, previous research has found that hypo- and hypercortisolism is associated with depression and anger36–39.\n\nOne explanation for the discrepancy in the variation of cortisol levels may be related to difference in perception and adaptation to stress by the caregiver, or even the course of the condition to a chronic state40. Leggett et al., in a study conducted with 164 caregivers of individuals with dementia, found patterns of hypocortisolism in populations with chronic stress, in mothers who take care of adolescents with autism or other serious mental disorders, or the parents of patients with cancer, but did not obtain the same patterns of cortisol variation in situations of non-chronicity41.\n\nIt is understood that the mechanism of maintenance of homeostasis in the body in response to the experiences of the individual throughout their life, is a dynamic process and involves interactions between biological and psychosocial factors in a way that does not always result in neuroendocrine adaptation, since the physiological response is dependent on the reaction of individuals to adverse situations. Therefore, the regulatory factors of a possible onset of hormonal disorder are variable, since they are influenced by the humoral state, individual experiences, social level, intellectual capacity, lifestyle, genetics of the individual, religiosity, age and even the biological material used to evaluate the analyte39,43,44.\n\nCortisol is not the only steroid that undergoes changes in response to stress. Other hormones, such as BDNF45, DHEA18 and progesterone46, interact and influence mood in individuals. There are studies that already suggest that the cortisol–DHEA ratio is a better marker for cognitive alterations than cortisol or DHEA alone, for example47. A previous study has shown that increased cortisol–DHEA ratios are related to the cognitive decline observed in caregivers of patients with dementia48, and this is not observed for cortisol alone.\n\nThe differences in the age groups of experimental samples, as suggested by the observed effects of age demonstrated by this and other work49,50 interfere with the circadian rhythm of cortisol. In this study, diurnal cortisol secretion followed a circadian rhythm, in line with normal conditions, in order to present higher levels of cortisol in the morning and decreasing levels throughout the day, for most cases. This is in accordance with the literature that predicts higher production of cortisol in the second half of the night, with a peak in the early hours of the morning and a progressive decrease during the day, with lower peaks during the first part of the night51. However, despite the positive correlation observed between the age of the caregiver and the level of cortisol in the afternoon, as well as in the mean cortisol of the three evaluated periods, cortisol changes with aging in humans are still considered controversial52.\n\nA limitation of our study was the lack of assessment of the coping strategies of caregivers. The majority of studies, including those with AD caregivers and chronic stress models, evaluate these strategies through the Coping Inventory for Stressful Situations (CISS)53,54. The CISS study is fundamental to understand how stress interferes in people's health in order to verify their ability to adapt to this disorder55,56, which could help to understand daily cortisol dosages within normality for most of the sample in research.\n\nOther limitations of the study were the absence of data on the length of stay of the caregivers with the elderly patients affected by AD, as well as the size of the sample in this study. These may have impaired the achievement of a significant correlation between the cortisol dosages evaluated and the other variables.\n\nIn conclusion, we found evidence that stress mechanisms are not necessarily linked to changes in the circadian rhythm of cortisol observed in caregivers of elderly individuals with AD. Therefore, we would suggest that this steroid hormone alone is not a reliable neuroendocrine marker for the disorder of depression. This evidence underscores the fact that the HPA axis does not have a standard relationship with exhaustive experiences and associated feelings of individuals. Taking into account past work10,40,48,57,58, these findings present a broader discussion, in terms of evidence, of cortisol responsiveness to caregiver health and possible co-influencers, such as the age and the impact of the CDR evolution of these individuals.\n\nIn future studies it is important to outline other possible cortisol modulators as well as the downstream neurosteroids. In addition, a more detailed study of the type of stress that reaches this group of caregivers, as well as their ability to adapt and face adversity in front of their work, should be included in the next analyses. This work is important from a physiological and psychological point of view, to understand the hormonal effects linked to depression in humans. In addition, this study indicates that the focus of the health of caregivers of patients with AD should be expanded and efforts to understand how routine can reflect on their body in form of disease should be made.\n\n\nData availability\n\nRaw data for this article are available on Open Science Framework (DOI: https://doi.org/10.17605/OSF.IO/DNTR7)59. Data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nConsent\n\nEach participant (patient and caregiver) gave written informed consent for inclusion in, and publication of, this study.", "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\nAcknowledgements\n\nThe authors would like to extend their gratitude to the Association of Studies, Research and Assistance to People with Alzheimer's Disease (AEPAPA) and to the Araucaria Foundation and Higher Education Personnel Improvement Coordination (CAPES).\n\n\nReferences\n\nAlzheimer's Association: 2013 Alzheimer's disease facts and figures. 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Data Source" }	[ { "id": "38013", "date": "19 Nov 2018", "name": "Helané Wahbeh", "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\nSummary:\nThis is an interesting study evaluating salivary cortisol levels in Alzheimer's patient caregivers with a correlational analysis of multiple measures with morning, afternoon, and evening cortisol values. There are a number of issues with the paper that are rectifiable and would strengthen it enough for indexing acceptance.\n\nAbstract:\nThis sentence in the abstract is very long and could be broken up for greater clarity: “Saliva samples were collected to assess the cortisol level of the caregivers three….caregivers and elderly patients.“\n\n\"this is not a good neuroendocrine marker of response to mood disorders\" This is a very strong conclusion that does not seem warranted from the results of the study and considering the vast literature on HPA axis measures and mood disorders.\n\n“dosage” Were they given cortisol? Maybe this should be \"values\"?\n\nIntroduction:\nChange “The main reason” to “One reason”.\n\nMethods:\nWhat time did participants wake up in relation to their first sample collection? What time did they go to sleep the night before? Was this a usual amount of sleep for them? Did you collect any information about exercise during the day, illness, and smoking? All of these affect cortisol levels and should also be accounted for.\n\nThe statistical analysis would be more powerful using a linear regression model with all the variables of interest rather than separate simple correlations. Even if you stick to simple correlations, you would need to include a multiple comparison correction.\n\nResults:\nIt would be helpful to put some of the demographic and/or questionnaire data into a table for easier readability.\n\nThe x-axis of Figures 1-4 is not clear. Is that showing time? Why are the numbers not in order? The figures are confusing and I’m not sure what relationship they are trying to show.\n\nDiscussion:\nThis is a very strong statement and not warranted considering the results of this one small study: “Our study showed that daily salivary cortisol levels did not differ significantly when associated with the level of depression in caregivers of patients with AD, suggesting that this is not a good marker of neuroendocrine response to mood disorders.”\n\n“possible onset of hormonal disorder” I don’t think you have to have a hormonal disorder to have dysregulated cortisol values.\n\nUnder limitations - the authors could also add no measurement of perceived stress. You only measured one day of cortisol and only three time-points. You could add pros and cons for different cortisol collection methods here (salivary vs blood vs urine), diurnal vs 24-hour collections.\n\n“Therefore, we would suggest that this steroid hormone alone is not a reliable neuroendocrine marker for the disorder of depression.” Again, this is a very strong statement considering the size and scope of the study and results.\n\nYou need to include a discussion of your results in relation to other papers that have evaluated cortisol in caregivers. Here are other papers I found with a quick search that you have not cited (de Vugt et al., 20051, Gallagher-Thompson et al., 20062 and Wahbeh et al., 20083). I am sure there are more.\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": "39244", "date": "18 Jan 2019", "name": "Maria F.B. Sousa", "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 addresses the related mechanisms between the hormonal systems of the hypothalamic–pituitary–adrenal axis, which produces cortisol. The study evaluated the relationship between the daily variation of salivary cortisol dosages and the level of stress in caregivers of patients with Alzheimer's disease (AD). It is an interesting article. However, some questions need to be clarified to improve the manuscript and make it clearer to the reader:\nAbstract:\n\nThe authors wrote in the ‘Conclusions’ section: “...the stress mechanism may be more complex and depend on more factors than the levels of this hormone”, so I suggest including in the ‘Conclusions’ section one or two factors to exemplify this.\nMethods:\nThere is this information that “In order to determine the size of the sample, we considered all patients that fit the inclusion criteria of the study and are serviced by AEPAPA”. However, the inclusion and exclusion criteria are not clear. I suggest writing about the criteria used in Association for Research and Assistance to Patients with Alzheimer’s (AEPAPA).\n\nThe authors concluded that “...the stress mechanism may be more complex and depend on more factors than the levels of this hormone”. So, I would like to know more information about the caregivers’ clinical comorbidities, because a clinical comorbidity can be an explanatory factor.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-672
https://f1000research.com/articles/7-671/v1	29 May 18	{ "type": "Research Article", "title": "Adequacy of food consumption in elderly Alzheimer’s disease in a community of Southern Brazil: a Cross-sectional study", "authors": [ "Glaucia Renee Hilgemberg", "Aline Jacoski de Oliveira Krüger da Silva", "Bárbara Luisa Fermino", "Camila Diedrich", "Simone Carla Benincá", "Débora Fernandes Pinheiro", "Flávia Ivanski", "Fernando Sluchensci dos Santos", "Weber Cláudio Francisco Nunes da Silva", "Caryna Eurich Mazur", "Roberta Fabbri", "Juliana Sartori Bonini", "Glaucia Renee Hilgemberg", "Aline Jacoski de Oliveira Krüger da Silva", "Bárbara Luisa Fermino", "Camila Diedrich", "Simone Carla Benincá", "Débora Fernandes Pinheiro", "Flávia Ivanski", "Fernando Sluchensci dos Santos", "Weber Cláudio Francisco Nunes da Silva", "Caryna Eurich Mazur", "Roberta Fabbri" ], "abstract": "Background: Alzheimer's disease (AD) is the most common cause of dementia, with a multifactorial etiology, in which the person has great difficulty identifying feelings of hunger, satiety, and feeding, which may affect their nutritional status. Pathologically, it is associated with neurodegeneration of synapses followed by neuronal loss, accompanied by glial proliferation surrounded by neurofibrillary tangles, beta-amyloid peptide (Aβ) deposition, inflammation and cerebrovascular injury hindering the ability to perform activities of daily living. This study aimed to analyze quantitatively the differences between an elderly group with AD and a control group, in terms of macro and micronutrient consumption evaluation. Methods: the study involved 69 participants who were assessed via collection of anthropometric measurements (weight, height and body mass index) with nutritional status being assessed by 24-hour food recall and three-day food record. Cognitive assessments were performed using the Mini-Mental State Examination (MMSE) and Clinical Dementia Ranting (CDR). Results: The intake of lipids in patients with severe dementia, was lower (p <0.05). The consumption of proteins showed a decrease with demential advance. For vitamins, there was a significant difference (p <0.05) in the amount of thiamine, niacin, vitamin D, E and K and calcium, chromium and iodine minerals, which were significantly reduced in AD patients (p <0.05). Conclusions: Decreases in macronutrient and micronutrient consumption may result in a consequent impairment of nutritional status, dementia progression, and decreased quality and life expectancy of elderly patients with AD.", "keywords": [ "Alzheimer’s Disease", "Food Intake", "Dementia", "Macronutrients", "Vitamins", "Cognition" ], "content": "Introduction\n\nAlzheimer's disease (AD) is the most common type of dementia, characterized by cognitive and neuropsychiatric manifestations that result in progressive disability1,2. Several factors contribute to the neurodegenerative process of AD. Lack of availability of certain nutrients, abnormal protein processing and neuronal membrane degeneration accelerate dysfunction and synaptic loss from the onset of disease3.\n\nThe medial temporal lobe is an area of the brain that is involved in the regulation of food intake4, and a site where neurodegeneration is typically present5. The behavioral modification of the AD patient interferes directly with the reduction of food consumption and the absorption of essential nutrients for the normal functioning of the body6.\n\nThe synthesis of synaptic membranes is dependent on various nutrients, such as folate, vitamin B12, vitamin B6, vitamin E, vitamin C and selenium. Inadequate ingestion and metabolism of these nutrients is related to increased phospholipid degradation and degeneration of neuronal membranes responsible for synapse loss3.\n\nThe nutritional status and food intake in the elderly differ by their cognitive, physical and biological capabilities. Taste reduction, difficulty in swallowing and xerostomia may be the result of neuropathological disorders, senility, or even induced by polypharmacy due to a higher incidence of chronic diseases resulting in them being more likely to be taking a combination of pharmaceuticals7,8.\n\nSince the nutritional status directly affects the patient's physiological condition, causing the elderly to be more susceptible to decreased biological function, making them more susceptible to falls, fractures, protein-energy malnutrition, and associated nutritional deficiencies6, this study had the intention of analyze the food intake in patients with AD, comparing it with the control group, identifying micro and macronutrient deficits that lead to worsening dementia and decreased quality of life.\n\n\nMethods\n\nThis study was approved by the Ethics Committee of the State University of Midwest (UNICENTRO) under number 026/2011. Patients and their caregivers received information on the purpose, methods, risks and benefits of research and gave written informed consent to participate in the study. Patients with any nutritional changes were referred to the Nutrition School Clinic of UNICENTRO for follow-up.\n\nA cross-sectional study with a control group was performed, with 37 elderly controls and 32 AD patients.\n\nThe elderly were attended and diagnosed clinically with AD in the health centers through the Unified Health System (SUS) and were passed through a confirmed diagnosis issued by a geriatric doctor or neurologist, and assisted by the Association of Studies, Research and Assistance to People with Alzheimer's (AEPAPA) in Guarapuava, Paraná.\n\nThe control group, in turn, was formed by healthy elderly participants of exercise groups and cultural activities for seniors, provided by the Social Welfare Department of the city of Guarapuava. A Mini-Mental State Examination (MMSE) was performed and evaluated by a trained healthy professional, applied to the health elderly as exclusion criteria for the control group. Also, they agreed in participate of the study and gave written informed consent in participate in the study.\n\nPatients and elderly without AD not found in their homes on three attempted visits on different days of the week, those who died, the residents of the countryside, and those who had moved to other cities, in addition to those unwilling or who rejected the survey after the first visit, along with those who did not agree with the terms of the Free and Informed Consent Term (TCLE) were excluded from the study.\n\nThe study occurred between August 2013 to June 2014, with an initial sample of 55 AD patients attending SUS in Guarapuava, and after applying the exclusion criteria, the sample consisted of 32 patients. The sample of healthy elderly was initially 50 participants, of which 13 declined to continue due to the amount of data that needed to collected and the time it would take to collect this data, consisting of 37 patients. All remaining individuals after applying exclusion criteria completed the study.\n\nTo evaluate the nutritional status, anthropometric measurements were collected: weight (kg), height (m), body mass index - BMI (kg/m2). Weight and height were collected according to the methods recommended by the Food and Nutrition Surveillance System - SISVAN9. When weighing and stature measurement was not possible due to the patient's health conditions, weight and height were estimated using theoretical formulas, using arm circumference (AC) and calf circumference (CC) measures, knee height and subscapular cutaneous fold. The formulae used to estimate weight were:\n\n\n\na) For men: weight = {[1.73 × Arm Circumference (cm)] + [0.98 × Calf Circumference (cm)] + [0.37 × subscapular skin fold (mm)] + [1,16 × Knee Height (cm)] = 81.69;\n\nb) For women: weight = {[0.98 × Arm Circumference (cm)] + [1.27 × Calf Circumference (cm)] + [0.4 × subscapular skin fold (mm)] + [0.87 × Knee Height (cm)] = 62.35}.\n\nThe formulae used for height estimation (m) in the elderly were:\n\n\n\na) For males: height = {64,19 - [0,04 × age (years)] + [2,02 × knee height (cm)]};\n\nb) For women: height = {84,88 - [0,24 × age (years)] + [1,83 × knee height (cm)]}.\n\nBy three-day and 24-hour dietary recall, the caregiver noted in forms all food and drink consumed (Forms are available as part of Dataset 110)). The collected information was used to perform a diet analysis including values of energy, macronutrients and micronutrients provided by DietWin® software, version 2008.\n\nAll values obtained from the average of four days of each nutrient were compared with the recommendations of the Dietary Reference Intakes (DRIs), according to the gender and age of the patient. As yet there are no specific recommendations for AD patients.\n\nEvaluated consumption in grams (g), milligrams (mg) and micrograms (mcg) of nutrients, and calculated the percentage of adequacy by the following formula: adjustment percentage = amount of ingested nutrient (g/mg/mcg)/DRI recommendation (g/mg/mcg) × 100.\n\nThe elderly were evaluated through the Mini Mental State Examination (MMSE) and Clinical Dementia Rating (CDR) between August 2013 to June 2014. Each category is classified based on scores - none/questionable dementia, mild dementia, moderate or severe11. The CDR and MMSE was applied and evaluated by a trained health professional.\n\nData were analyzed using the SPSS 20.0 statistical software for Windows, presented as median and interquartile range, relative and absolute frequency. Prior to the analysis, the following assumptions were tested on the numerical variables: homogeneity of the variances using the Levene test, and distribution of the data using the Shapiro-Wilk test. If the assumptions were incorrect data was analyzed using the Kruskal-Wallis test. When necessary, we used the multiple comparisons analysis with the Bonferroni level of significance correction. The Chi-square test, Fisher's Continuity Correction and Exact Correction were used to identify associations between the diagnosis of AD and clinical and sociodemographic variables. A significance level of p <0.05 was adopted.\n\n\nResults\n\nIn total, 69 participants took part in the study: 37 from the control group and 32 from the AD group.\n\nTable 1 shows anthropometric characteristics of the sample, these data are characterizing this population according to CDR, age, current weight, height and BMI. Data are presented as median (P25-P75).\n\nGuarapuava, PR, Brazil. 2013.\n\nData are presented as median (P25-P75) CDR: clinical dementia rating (cases); BMI: Body Mass Index; n: sample number.\n\nThe age of the elderly ranged from 57 to 94 years, with a median of 74 years, with a median height of 1.58m of the total sample. Regarding body weight, the control group had the highest average, and the weight in AD patients decreased with increased severity of disease.\n\nRegarding the sociodemographic characteristics (Table 2), 45 (65.2%) were female; 20 were married in the control group (29.0%) and in the AD group there were 7 (10.1%); 3 from the control group were unmarried (4.3%) and in the AD group there were 7 (10.0%). There were 13 widowers in the control group (18.8%), and 17 (24.5%) in the AD group.\n\nGuarapuava, PR, Brazil. 2013.\n\nCDR: clinical dementia rating (cases); n: sample number.\n\nThe average family income reported by the participants in the control group was R$ (Brazilian real - BRL) 904.00 ± 8.99 (US$ 275.76) and the participants with the disease presented income around R$ 1.031.13 ± 67.29 (US$ 314.54).\n\nTable 3 shows the comparison of carbohydrate intake. The consumption was lower in the control group but there was no difference between the groups of patients with dementia (p> 0.05). In contrast, with respect to the consumption of proteins, as disease progressed this concomitantly decreases. The only macronutrient that gave a significant difference (p <0.05) was the lipid values, in relation to the control group (113.64% adequacy), which presented a greater consumption than CDR-1, mild dementia (86.95% adequacy) and CDR-3, severe dementia (7751% adequacy).\n\nGuarapuava, PR, Brazil. 2013.\n\nData are presented as median (p25-P75) CDR: clinical dementia rating (cases); Kruskall-Waliss (Bonferroni for multiple comparisons). * There was a statistical difference between elderly control subjects and elderly with mild and advanced dementia (p<0.05). ‡There was no difference between groups (p>0.05).\n\nTable 4 presents the data in relation to the intake of vitamins. For vitamin B1, the control group consumed a median 0.77 (0.55-0.97) mg on median, lower than that of CDR-2, which consumed a median of 1.27 (1.1-1.47) mg (p <0.05). For vitamin B3, differences were also observed: the median control group ingestion was 6.22 (4.85-8.68) mg. CDR-2 and CDR-3 patients had a significant higher (p <0.05) consumption (10.27 (8.8-14.05) and 11.22 (9.04-14.1) mg, respectively). Vitamin D, E, and K intake were higher in the control group when compared to all stages of dementia (vitamin D, p> 0.05; vitamin E, p> 0.05; vitamin K, p <0.05).\n\nAmong the minerals shown in Table 5, only calcium, chromium and iodine showed differences between the groups (p <0.05).\n\nGuarapuava, PR, Brazil. 2013.\n\nData are presented as median (P25-P75). CDR: clinical dementia rating (cases); n; sample number; Kruskall-Waliss (Bonferroni for multiple comparisons). There was a statistical difference between elderly control subjects and elderly with moderate dementia (p<0.05). * There was a statistical difference between elderly control subjects and elderly with mild and advanced dementia (p<0.05). † There was a statistical difference between elderly control subjects and elderly with mild, moderate and advanced dementia (p<0.05). ‡ There was no difference between groups (p>0.05).\n\nGuarapuava, PR, Brazil. 2013.\n\nData are presented as median (P25-P75). CDR: clinical dementia rating (cases); n; sample number; Kruskall-Waliss (Bonferroni for multiple comparisons). There was a statistical difference between elderly control subjects and elderly with moderate dementia (p<0.05). * There was a statistical difference between elderly control subjects and elderly with mild and advanced dementia (p<0.05). † There was a statistical difference between elderly control subjects and elderly with mild, moderate and advanced dementia (p<0.05). ‡ There was no difference between groups (p>0.05).\n\n\nDiscussion\n\nDiet and nutritional factors are known modifiable factors for dementia and cognitive decline later in life, with significant associations between an inadequate nutritional status and behavioral disorders with greater impact on activities of daily living12.\n\nThe sample was made up mostly of patients with advanced dementia (50% of AD patients), and is similar to the study of Goes et al.7 where 40% of the sample were in the advanced stages of the disease. Advanced disease is characterized by severe memory loss, with no capacity to make decisions, and requires a caregiver or person who can assist in the practice of activities of daily living, and most of them can result in food imbalances and consequent malnutrition.\n\nA high BMI in middle age is associated with an increased risk for the development of dementia7. The elderly studied in the control group, and in the CDR1 and CDR2 groups, were overweight and as the staging of the disease increases, BMI decreases. This overweight condition is likely related to the food preferences found for this group, carbohydrates and lipids. These preferences and dietary deficiencies could be caused by the low family income found in the study, and also due to the decrease in executive functionality and dysphagia3, changing the consumption of proteins for carbohydrates13.\n\nTieland et al.12, in the Netherlands, analyzed three groups of elderly people, 707 individuals living independently in the community, of which 194 were fragile and 276 were institutionalized. The results showed that 35% of the institutionalized elderly had inadequate protein intake, below 0.7g/kg/day. As protein intake is impaired, there is an accelerated loss of muscle mass, sarcopenia13, low hemoglobin levels, and a consequent increase in the mortality rate14.\n\nAccording to IBGE research15 held in Brazil, the average consumption of lipids for men and women 60 years or more, is approximately 49 grams. In the present study, the control group had a higher intake, at 42.89 grams, in comparison to CDR 3, 32.24 grams. Diets high in fat contribute significantly in β-amyloid accumulation, tau hyperphosphorylation and an inflammatory state in the peripheral organs and brain16.\n\nAnalyzing the thiamine intake (vitamin B1), differences in consumption between groups can be seen. The control group had the lowest intake, likely related to the vitamin supplementation initiated after diagnosis of dementia. Relating IBGE data15, the micronutrient ingestion for elderly is approximately 1.05 mg, while the amount for the AD group was far below.\n\nIn the study by Morris et al.17, the total of 3,718 participants aged 65 years and over had dietary data taken, with at least two evaluations and analyzes of cognitive changes over a median of 5.5 years. In this prospective study, nutritional deficiencies of niacin (vitamin B3) were associated with AD and cognitive decline. Since the higher intake of vitamin B3 was associated with a slow rate of cognitive decline and its retardation. The nutritional recommendation for the elderly aged 60 years or more is the intake of approximately 22.7 mg/day7.\n\nThere are studies in which the importance of vitamin D is postulated in cases of dementia18,19 as it promotes the regulation of calcium homeostasis, β-amyloid peptide clearance, slows down and/or improves cognitive decline, and has an important antioxidant and anti-inflammatory effect20. In the cohort study conducted by Karakis et al.18, in the USA, the final sample being composed of 1663 patients, vitamin D was associated with lower hippocampal volumes, helping to prevent brain lesions and cognitive decline. The ingestion of calciferol in the related study was impaired in all dementia phases. This may be due to their eating habits. The recommendation according to IBGE15 for individuals over 60 years or more is on average 3.05 mcg/day.\n\nVitamin E eliminates free radicals, with some chemical forms have a potent anti-inflammatory effect, and the α-tocotrienol congener protects the cortical and hippocampal neurons from apoptosis. Vitamin E deficiency in human manifests primarily as peripheral sensory neuropathy, which demonstrates its essential role in the formation and maintenance of CNS function, supporting its potential use in the prevention and treatment of neurodegenerative diseases21. According to the recommendations of the National Institutes of Health of the United States, the recommended daily dose of vitamin E for an adult is 15mg22. For the IBGE15, 3.8 mg/day is recommended for elderly individuals.\n\nA study in the Netherlands23 involved a sample of 5395 participants over 55 years of age who did not present with dementia. These individuals completed checklists about their eating habits over the past year. The indicated food would have been consumed at least twice a month. Participants were followed for 9.6 years. Of these, 465 developed dementias, of which 365 were diagnosed as having AD. Individuals with vitamin E consumption had a 25% lower risk of developing dementia. It is interesting to analyze the control group of the present study, because it was observed that when comparing the group without AD, all the dementia groups had a lower consumption of this important vitamin.\n\nHowever, with regard to vitamin K, studies demonstrate the importance of activities related to the brain24,25. It is related to the transport of the apolipoprotein E (ApoE), a major of marker26,27. In the study of Carrié et al.28, conducted with mice to analyze the cognitive ability of a diet low in vitamin K, a vitamin K depleted diet resulted in higher cognitive deficits. In the study by Presse et al.24, a paired control case study of 31 control subjects and with AD, respectively, as in the present study, the healthy group consumed more food containing this vitamin.\n\nThe number of works that present calcium as an important underlying factor in the process functional changes of aging, especially in AD has increased29,30. Its dysregulation causes loss of function and mutations in the protein presenilin 1 (PS1), which is a risk factor for the onset of the disease. Other effects include alterations in the autophagic and lysosomal pathways31,32, increased oxidative stress and consequent inflammation in neuronal cells33,34. Patients without neurodegeneration, however, consumed less calcium in their diet compared to those who had moderate dementia, CDR-2 and advanced dementia, CDR-3. The increased intake of dairy products in the diet of survey participants with dementia due to preference from soft textures of the food at this stage of life and stage of disease is the likely cause of this finding.\n\nAmong the micronutrients observed in Table 5, we highlight the chromium, which was less consumed by AD patients in CDR-3, as recommended by IBGE17. In the study by Krikorian et al.34, a double-blind study was conducted in Cincinnati, USA, to evaluate whether chromium supplementation helps in memory and neuronal function in the elderly with cognitive decline. The results were that chromium picolinate supplementation may significantly improve cognitive inhibitory control and brain function in individuals who have neurodegeneration.\n\nA cross-sectional study was conducted in Brazil in a group of 135 elderly women with mean age of 68 years, in which 42% of the women analyzed had iodine deficiency35. Iodine deficiency causes thyroid dysfunction, as well as hypertension, dyslipidemia, muscle wasting, frailty and neuromuscular dysfunction, reducing quality and life expectancy36. There is still no daily recommendation for iodine intake specifically for the elderly population.\n\nThe sample size and the fact that the values presented being an estimative from the nutritional consumption were the limitations of the study. Therefore, these results are in agreement with similar researches that consider nutritional consumption7.\n\n\nConclusion\n\nThe nutritional status of an individual is associated with cognitive health and disease progression. According to the results, it is concluded that the progression of dementia is associated with lower levels of dietary intake of some macro and micronutrients. Among the macros, reduced intake of lipids in mild and severe dementia can be observed. Regarding the micronutrients vitamin D intake showed a reduction in mild dementia stage. The vitamins B5, E, and K, and minerals calcium and chromium, had reduced consumption in severe dementia cases. Specific nutritional interventions can alter the development of the disease with low risk of side effects and with satisfactory results particularly in the early stages of the disease.\n\n\nData availability\n\nAll raw data was freely available on Open Science Framework site. Dataset 1: Adequacy of food consumption in elderly Alzheimer’s disease in a community of Southern Brazil: a Cross-sectional study. http://doi.org/10.17605/OSF.IO/89USV10\n\nThe data is available under a CC0 1.0 license.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors would like to thank the Araucaria Foundation, National Council for Scientific and Technological Development (CNPq), Coordination of Improvement of Higher Level Personnel (CAPES) and AEPAPA for providing funding for study.\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\nSanchez-Mut JV, Aso E, Panayotis N, et al.: DNA methylation map of mouse and human brain identifies target genes in Alzheimer’s disease. Brain. 2013; 136(Pt 10): 3018–27. 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PubMed Abstract \| Publisher Full Text\n\nSegev Y, Livne A, Mints M, et al.: Concurrence of High Fat Diet and APOE Gene Induces Allele Specific Metabolic and Mental Stress Changes in a Mouse Model of Alzheimer’s Disease. Front Behav Neurosci. 2016; 10: 170. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarrié I, Bélanger E, Portoukalian J, et al.: Lifelong low-phylloquinone intake is associated with cognitive impairments in old rats. J Nutr. 2011; 141(8): 1495–501. PubMed Abstract \| Publisher Full Text\n\nFrazier HN, Maimaiti S, Anderson KL, et al.: Calcium’s role as nuanced modulator of cellular physiology in the brain. Biochem Biophys Res Commun. 2017; 483(4): 981–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOverk CR, Rockenstein E, Florio J, et al.: Differential calcium alterations in animal models of neurodegenerative disease: Reversal by FK506. Neuroscience. 2015; 310: 549–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcBrayer M, Nixon RA: Lysosome and calcium dysregulation in Alzheimer’s disease: partners in crime. Biochem Soc Trans. 2013; 41(6): 1495–502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee JH, McBrayer MK, Wolfe DM, et al.: Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification. Cell Rep. 2015; 12(9): 1430–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSupnet C, Bezprozvanny I: Neuronal calcium signaling, mitochondrial dysfunction, and Alzheimer’s disease. J Alzheimers Dis. 2010; 20 Suppl 2: S487–98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrikorian R, Eliassen JC, Boespflug EL, et al.: Improved cognitive-cerebral function in older adults with chromium supplementation. Nutr Neurosci. 2010; 13(3): 116–22. PubMed Abstract \| Publisher Full Text\n\nDestefani SA, Corrente JE, Paiva SA, et al.: Prevalence of iodine intake inadequacy in elderly Brazilian women. A cross-sectional study. J Nutr Health Aging. 2015; 19(2): 137–40. PubMed Abstract \| Publisher Full Text\n\nBoelaert K: Thyroid dysfunction in the elderly. Nat Rev Endocrinol. 2013; 9(4): 194–204. PubMed Abstract \| Publisher Full Text" }	[ { "id": "38377", "date": "12 Nov 2018", "name": "Deborah R. Gustafson", "expertise": [ "Reviewer Expertise Nutritional", "dementia and aging 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\nThis manuscript describes the dietary intake and anthropometric measures of 32 AD cases and 37 controls in southern Brazil. The hypothesis is sound regarding changes and differences in both diet and anthropometric measures in AD and related disorders. In addition, there is limited knowledge of this in Brazilian samples of elderly, an underrepresented group. Despite the strengths of the research question, the reviewer has several questions.\nThe cited prevalence of AD is confounded by understanding whether the authors refer to a clinical vs neuropathological diagnosis of AD. Since there are no brain imaging data referred to nor presented, it is assumed that the AD cases referred to and included in this study are based on clinical diagnostic criteria only.\n\nDietWin (a 10 year old version) is the dietary analysis software cited in the Methods section. It is unclear as to how the nutrient intake data is estimated, and whether it is appropriate for southern Brazil. While the reviewer looked to the DietWin software website, noting its apparent Brazilian application, additional details regarding DietWin would be appreciated since the nutritional comparisons are based entirely on its output. Other details of dietary/food intake data entry and analysis are missing.\n\nA 3 day dietary intake was merged with one 24h dietary recall and an average across 4 days was calculated. These are 2 very different methods of acquiring dietary intake data, such that adding a 24h recall to a 3 day dietary recall is not 'summing' the same thing.\n\nThe case group, AD patients, is being compared to a very healthy (& younger) control group. The control group does not appear to be a worthy comparison, yet they are comprised of 'patients'. Use of the latter term is incorrect.\n\nIt is stated that, 'By three-day and 24-hour dietary recall, the caregiver noted in forms all food and drink consumed'. Does this mean that caregivers of BOTH AD cases and controls provided information on dietary intake, or only for those who needed help. This is differential depending on the need of the participant and could introduce a bias. It is unclear what proportion of the sample self-reported vs had a caregiver report dietary intake. This very healthy control group would not seem to need a caregiver. Secondary reporting of dietary intake data is notoriously inaccurate. In addition, it is anticipated that the cases were more likely to have a caregiver report on their behalf.\n\nIt is unclear what proportion of participants required estimation of anthropometry using the equations provided based on measures not requiring standing. Differences in measurement also may introduce a bias.\n\nGiven the small sample size, the results and discussion are speculative at best.\n\nAn English language edit is required.\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": "44054", "date": "06 Feb 2019", "name": "Charlotte E. Teunissen", "expertise": [ "Reviewer Expertise Alzheimers disease", "biomarkers" ], "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 paper should provide a clear problem statement and literature review to provide a rationale for the study. There is no overview of the literature of previous similar studies, and the added value of the current study is thus not clear.\nThe method to perform a 24 food recall and just 3 days food record is likely way too short to get an impression of food intake of patients, which can vary over weeks. The use of this methodology is not sustained. And since this methodology does not seem appropriate, I judge that the conclusions cannot be drawn.\nThe discussion mixes concepts: data obtained in longitudinal risk and epidemiological studies in pre-dementia patients address a different subject than the current study trying to study the cross sectional food intake in established dementia patients.\n\nAlzheimer disease should be diagnosed using state of the art criteria (e.g. including biomarkers, see Jack et al Neurlology 2018)\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?\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? No", "responses": [] } ]	1	https://f1000research.com/articles/7-671
https://f1000research.com/articles/7-670/v1	29 May 18	{ "type": "Research Article", "title": "A retrospective review on minimally invasive technique via endoscopic thoracic sympathectomy (ETS) in the treatment of severe primary hyperhidrosis: Experiences from the National Heart Institute, Malaysia", "authors": [ "Ahmad Farouk Musa", "Vignaa Prashanth Gandhi", "Jeswant Dillon", "Rusli Bin Nordin", "Vignaa Prashanth Gandhi", "Jeswant Dillon", "Rusli Bin Nordin" ], "abstract": "Background: Hyperhidrosis is due to the hyperactive autonomic stimulation of the sweat glands in response to stress. Primary hyperhidrosis is a common yet psychologically disabling condition. This study will describe our experience in managing hyperhidrosis via endoscopic thoracic sympathectomy (ETS). Methods: The information was obtained from the patient records from 1st January 2011 until 31st December 2016. Pertinent information was extracted and keyed into a study proforma. Results: 150 patients were operated on but only 118 patients were included in this study. The mean age was 22.9±7.3 years. The majority (54.2%) had palmar-plantar hyperhidrosis and 39.8% had associated axillary hyperhidrosis. Excision of the sympathetic nerve chain and ganglia were the main surgical technique with the majority (55.9%) at T2-T3 level. Mean ETS procedure time was 46.6±14.29 minutes with no conversion. Surgical complications were minimal and no Horner’s Syndrome reported. Mean hospital stay was 3.5±1.05 days. The majority of patients (67.8%) had only one follow-up and only half of the study sample (58.5%) complained mild to moderate degree of compensatory sweating, even though the long-term resolution is yet to be determined by another study. Following ETS, 98.3% of patients had instant relief and resolved their palmar hyperhidrosis. Predictors of CS were sympathectomy level and follow-up. The odds of reporting CS was 2.87 times in patients undergoing ETS at the T2-T3 level compared to those undergoing ETS at the T2-T4 level. The odds of reporting CS was 13.56 times in patients having more than one follow-up compared to those having only one follow-up. Conclusion: We conclude that ETS is a safe, effective and aesthetically remarkable procedure for the treatment of primary hyperhidrosis with only half of the patients developing mild to moderate degree of CS. Significant predictors of CS were sympathectomy level during ETS and frequency of follow-up after ETS.", "keywords": [ "Primary hyperhidrosis", "Endoscopic thoracic sympathectomy", "Compensatory sweating", "National Heart Institute" ], "content": "Introduction\n\nHyperhidrosis is a pathological condition of excessive sweating beyond the physiological needs for thermoregulation, which may severely disrupt a person’s quality of life and make their social interaction difficult1,2. Primary hyperhidrosis results from the overactive sudomotor system which controls sweat output with no apparent cause2. And it is important to differentiate this condition from secondary hyperhidrosis, which can be due to many conditions including endocrine disorders, infection, genetic, malignancy, neurologic and miscellaneous causes3.\n\nEpidemiological studies in the United States4,5 indicated that the prevalence of hyperhidrosis ranged from 0.6% to 9% in the particular populations studied; indicating this disease is not a rare event. It is interesting to note that the prevalence outside the United States are significantly higher and vary extensively. The observed prevalence of primary hyperhidrosis is 13.9% in Japan, 14.5% in China, 16.3% in Germany, 16.7% in Canada and 20.3% in Sweden6–9. And over the past several decades, different researchers10–13 have speculated positive family histories in patients with primary hyperhidrosis, widely ranging from 5% to 50%; however, there was no substantial data being offered until recently. Ro et al.14 also reported in 2002 that patients with hyperhidrosis had 65% genetic prevalence of autosomal dominance with variable penetrance and with no evidence of sex-linked transmission. This means that a child of a parent with hyperhidrosis has a 25% chance of developing hyperhidrosis due to the likelihood of phenotypic expression of 0.28. It is also important to note that there is potential genetic linkage to chromosome 143.\n\nThere are important anatomic points that need to be understood as they relate to the main intervention discussed in this paper; however, it should be understood that the neuroanatomy of the sweating mechanism is complex beyond the scope of this paper. In relation to hyperhidrosis, the anatomy involves nerve fibers that originate from the hypothalamic preoptic sweat center to the spinal cord where they meet and synapse with the preganglionic fibers. These fibers then leave the spinal cord coursing along the ventral nerve roots and terminate in the sympathetic ganglia15,16. In the sympathetic ganglion, pre- and postganglionic fibers synapse, which then track peripherally and end at their target organs, in this case, the sweat glands16 (Figure 1).\n\nReprinted from Thoracic Surgery Clinics, Volume 18, Issue 2, Shargall Y, Spratt E, Zeldin RA. Hyperhidrosis: What is it and why does it occur? Pages 125–132, Copyright (2008) with permission from Elsevier.\n\nThe pathophysiology of the underlying hyperhidrosis is intricate and not fully understood, although, it is commonly believed to stem from an exaggerated central response to normal emotional stress or environmental stimuli. In most cases, it occurs intermittently and spontaneously. And it should not be considered as a psychiatric disorder17. In primary palmar hyperhidrosis, there is a hyper-functioning emotional element of the central sudomotor nervous system, substantiated by the observation that excessive sweating does not transpire during sleep and is aggravated by emotional stimuli. The body undergoes a number of physiological changes during mental stress such as increased skin sympathetic nerve activity, excessive sweating, pronounced vasoconstriction and a concomitant increase in evaporation jointly termed as “cold and clammy” hands18,19.\n\nWhile the treatment of primary hyperhidrosis could be medical or surgical, this paper will focus on the surgical aspect of minimally invasive surgery known as endoscopic thoracic sympathectomy (ETS). Most ETS procedures are done bilaterally, with denervation of the sympathetic chain on both sides, and hence, the lungs have to be deflated in order to reach the sympathetic chain. Single lung anesthesia is accomplished by the placement of a suitable sized single or double lumen endotracheal tube. Most literatures20–24 describe the use of a double lumen tube rather than single, which is what our center practiced, as it is easier to achieve a single lung separation. At our center, two incisions are made for thoracoscopic access and ports with lumens. The skin incisions, of about 15mm, are made around the mid-axillary line over roughly the fifth intercostal space. After the underlying lungs are collapsed on one side, the pleural space is entered by blunt dissection. A 5 or 10mm 0-degree thoracoscope is introduced into the cavity. Under endoscopic guidance, a second “stab” incision is made near the sub-mammary fold.\n\nThe upper thoracic sympathetic trunk is located underneath the pleura as it traverses over the neck of the ribs. It can be usually confirmed by passing a probe along the neck of the rib and feeling the string-like solid trunk. The cervicothoracic (stellate) ganglion is obscured as it lies beneath a distinctive fat pad on the neck of the first rib. The first and the second rib can be distinguished by the location of the supreme intercostal artery and by the curvature of the rib25. There is some controversy in regards to reaching a consensus as to which level of the ganglion (T2–T4) should be resected to achieve best results. The sympathetic chain is then resected, transected or ablated with a cautery, or divided with a harmonic scalpel or a clip is used, depending on the center and surgeon performing the surgery, though we at the Institute practiced thermo-resection and stripping. It is vital to identify and divide the Nerve of Kuntz. But cauterizing the trunk above the T2 ganglion should be done with caution as there is a risk of causing Horner’s syndrome, characterized by miosis, partial ptosis, anhidrosis, and enophthalmus, which is a disastrous and avoidable complication26. After completing the surgery, an apical chest drain is placed at the anterior wound and the lung is re-inflated. And the procedure is then repeated on the other side.\n\nIt is interesting to note, at this stage, based on an analysis of forty-two different ETS techniques by Kopelman and Hashmonai27, there were no distinct differences shown by the different techniques; if the correct level of interruption is achieved, the results are excellent and reproducible. The most imperative notion about nerve disruption is that there is adequate separation between the ends of the chain so that regeneration is halted. Although resection yields better results in terms of lower recurrence rates, most ETS procedures prefer ganglionic thermo-ablation or thermo-transection of the chain due to the relative simplicity of the latter rather than endoscopic resection28–30.\n\nAlthough ETS is a minimally invasive procedure aimed at improving the quality of life, it is not without complications. The main side effects of ETS for hyperhidrosis are compensatory sweating (CS), bradycardia, and Horner’s syndrome. Chou et al. comments that generally, “the higher the level of interruption on the chain, the higher is the expected regret rate”31. Horner’s syndrome is an undesirable complication, with its occurrence following surgery ranging widely from 0% to 40%32–34. The rate of this complication occurring is higher in patients who undergo ETS for facial hyperhidrosis, as injury by electrocautery, thermo-ablation, traction, or surrounding inflammation can occur due to improper identification of the second rib. Phantom sweating and gustatory sweating are two side effects of ETS and the etiology of which still remains obscure35,36. The former is a sensation of sweating in the target area after sympathectomy without any physical evidence of sweating, whereas the latter is mainly sweating on the face triggered by spicy and some other foods. Bradycardia has also been noted in several studies37,38 after ETS for hyperhidrosis.\n\nCS is by far the most troublesome and disagreeable complication of ETS, producing subjective and objectively quantifiable increased sweating in body segments usually below the area of denervation such as the torso, groin, and lower extremities. However, the wide disparity of the occurrence of CS displayed in the literature clearly highlights the lack of standardization in reporting data. It is mindboggling that the exact same procedure would have such a huge difference of CS rates as it ranges from 3% to 98%39,40. Due to this unpredictable factor, all patients should be told preemptively of this possible complication and that it is irreversible41. The pathophysiology of CS remains unknown and has not been fully described by the literature. Available data regarding the pathophysiology of CS is not fully verified, hence, the mechanism of CS is said to be multifactorial and, thus, difficult to quantify31.\n\n\nMethods\n\nThis is a single center retrospective review of patients who underwent ETS for primary hyperhidrosis at the National Heart Institute (IJN), Malaysia. Patients’ medical records from 1st January 2011 until 31st December 2015 were reviewed in a retrospective manner to identify the success rate of the surgery, occurrence rate of CS and also identify factors that might influence the rate of CS post ETS.\n\nEthical approval was obtained through the National Heart Institute Research Ethics Committee (IJNREC 206/2017). No amendment was made throughout the duration of the study. This study was also registered with the National Medical Research Register (NMRR-18-29-39863, Ministry of Health, Malaysia on the 12th February 2018.\n\nThe main aim of the research was to evaluate the efficacy of ETS, provide long-term follow-up data and investigate the occurrence, severity and significant predictors of CS at the National Heart Institute (IJN).\n\nA number of potential pre-operative, intra-operative and post-operative variables were collected after a thorough literature review. The list of variables was reviewed and scrutinized multiple times prior to the start of data collection, taking into account professional advices and availability of certain information in existing medical records in this particular center. The list of variables was then compiled and structured into a proforma (Supplementary File 1), which was used as our data collection sheet in this study.\n\nEvery patient who had ETS in IJN from 1st January 2011 to 31st December 2015 was included in this study. All patients who had ETS for primary hyperhidrosis only were accounted for in this particular study. No patient underwent two ETS surgeries in IJN during this study period. The exclusion criteria for this study were patients who had primary hyperhidrosis, and having other medical conditions that cause hyperhidrosis (such as hyperhidrosis secondary to hyperthyroidism). All patient medical records had to be complete, and most importantly the follow up reports. Patients who defaulted follow up were not included in this study as it defeats the main aim of this study, which is to determine the rate of CS and success rate of ETS.\n\nStatistical analysis was done using the IBM SPSS Version 24.0. Continuous data were analyzed and depicted as means and standard deviations whereas categorical data were reported as frequencies and percentages. Chi Square and Fisher Exact tests were used to test the association between categorical variables. Differences were considered significant if p<0.05. Following a univariate binary logistic regression analysis for predicting CS, independent variables with p values of less than 0.25, were selected for entry into the multivariable analysis after checking for possible multicollinearity and interactions. In the multivariate binary logistic regression analyses, significant and independent predictors of CS were modeled using the stepwise method for the preliminary final model after checking for model fitness using three goodness-of-fit assessments: Hosmer-Lemeshow test, Classification Table, and area under the curve (AUC) of the receiver operating characteristics (ROC) curve. Based on these assessments, the final prediction model of CS will be identified. The percentage of variance explained by the final model will be indicated by two pseudo R square measures: Cox & Snell R square and the Nagelkerke R square.\n\n\nResults\n\nWe operated on 150 patients from January 2011 to December 2015. However only 127 patients fit into the inclusion criteria with complete case records. Nine patients were then excluded in accordance to the exclusion criteria for not attending follow-up. The rest of the 118 patients were included for data analysis (Table 1). The patients in this particular study sample demonstrated an almost equal distribution of male and female gender, with slightly a higher proportion of males (57.6%) having ETS compared to females (42.4%). The mean age of having ETS was 22.91 ± 7.3 years, the youngest being nine years old whilst the eldest was aged 52 years. The majority of the patients who had ETS were aged 21–30 years, representing 44.9% of the sample whereas patients aged 31 years and above represented the lowest proportion (11.9%) of the study sample.\n\n* Six missing data for obese category (3 were excluded based on the location of their compensatory sweating and 3 due to loss to follow up), BMI: body mass index\n\nThe ethnic distribution of our study corresponds to the racial distribution of the Malaysian population (Table 1). Malay was the dominant ethnic group (79.7%), followed by Chinese (13.6%) and Indian (6.8%). The pre-operative body mass index (BMI) distribution is as follows: the majority (48.2%) of patients was in the normal category, followed by overweight (33.9%), underweight (9.8%), and obese category (8.0%).\n\nAll 118 patients had more than two areas affected by primary hyperhidrosis, hence they are grouped into areas that affected them the most (Figure 2). Out of the 118 patients, 64 had primary hyperhidrosis affecting their hands and feet, which accounts for the majority of the sample (54.2%). Patients who had the disease affecting their axilla and on top of their hands and feet accounted for 39.8% of the sample. The most severe presentation of primary hyperhidrosis, which affects more than three regions (hands, feet, axilla and the face) only totaled seven patients (5.9%). It is also important to note that one patient had isolated primary hyperhidrosis on the hands, but was lost to follow up, hence not included in this study.\n\nThere were very little variations to the surgery that had been performed, as we were looking at a single center performing a state of the art surgery. The technique used by all surgeons was thermo-resection and stripping. The level of sympathectomy that was predominantly used in this sample was T2-T3 accounting for 55.9% of the total surgeries (Table 2). This is followed by the T2, T3, and T4 level, accounting for 41.5% of the surgeries. Three patients had sympathectomy at levels T3-T4 that only contributed 2.5% to the total surgeries. All surgeries in IJN were performed in a similar manner with exceptions to the level of sympathectomy. The mean duration of surgery was 46.6 ± 14.29 minutes. Impressively, the mean duration of stay in the hospital after ETS was only 3.5 ± 1.05 days.\n\nIn IJN, the number of patients experiencing any complications operatively and post- operatively was staggeringly low. The majority of the patients (89.1%) who underwent ETS had no complication during surgery or post operatively (Table 3). Seven (5.9%) patients developed pneumothorax as a result of the surgery and four (3.4%) patients had severe pain at the operation site. One patient developed bradycardia, a relatively rare complication resulting from sympathectomy of the thoracic ganglia. Interestingly, one patient developed post-sympathetic neuralgia, which is incapacitating due to the constant pain and interferes with the patient’s daily life but none developed Horner’s syndrome.\n\nAll of our patients who had ETS had at least one follow-up post-ETS. In IJN, 67.8%, 24.6%, and 7.6% of the patients had one, two, or three follow-up, respectively. Our primary aim was to look at the resolution of palmar hyperhidrosis after ETS. In IJN, the outcomes were excellent as 98.3% of our patients had almost complete (90–100%) resolution of palmar hyperhidrosis. One patient had partial resolution of symptoms and one more patient had no change in the palmar hyperhidrosis.\n\nWe also noted the degree of resolution in other regions, such as the axilla and plantar areas. Only 6 (5.1%) patients had axillary and plantar hyperhidrosis resolution post ETS whereas the majority of the patients had partial resolutions of symptoms only (Table 4). We noticed that 35.6% of patients with hyperhidrosis involving the axillary region had partial (60–90%) resolution of symptoms and 61.9% of patients from the plantar group had partial resolution of their primary hyperhidrosis. It also important to highlight that 17.8% of the patients from our center who had ETS did not have any changes in the degree of sweating at their plantar region.\n\nCS is a pertinent factor whenever ETS is performed for primary hyperhidrosis. It is one of the major factors that have always influenced the decision as to whether ETS should be done or not. In our study, the rate of CS was 58.5% (Figure 3).\n\nOf those who developed CS, 52.2% reported the mild form of CS (Figure 4). It means that the majority of patients experienced a much more tolerable form of CS compared to their palmar hyperhidrosis. Only 15.9% of those who developed CS reported the severe form CS and described the severity and location of the sweating as worse than their initial condition. Some also regretted their decision to have the surgery.\n\nThere are many areas that may be affected by CS post-ETS. These areas have been grouped according to their frequency. The area that was most affected by CS was the back (29.0%) whilst the least being the chest and face (2.9%). There appears to be a certain predilection to the combination of areas where CS affected the most. CS occurring at the back and back of thighs was as high as 18.8% followed by 14.5% at the trunk and back of thighs. Other areas affected by CS post-ETS are shown in Table 5 below.\n\nPatients who had CS and who had at least two follow-ups were also asked regarding the progression of CS. They were grouped into three categories: reducing, increasing or status quo. Out of the 69 patients who reported CS, the majority (88.4%) reported that their CS remained the same in their later follow-ups post ETS. Four (5.8%) patients claimed that their CS worsened over time and four (5.8%) patients claimed that their CS improved over time.\n\nA Chi-Square and Fisher exact test of independence was performed to see if there is an association between age, ethnicity, gender, BMI and CS among 118 IJN patients (Table 6). No significant association was observed between all four patient characteristics and CS.\n\nχ2= Chi-square value, df=degree of freedom, aFisher exact test, bSix data missing from obese category, Significant at p<0.05, BMI: body mass index\n\nTo determine the association between location of primary hyperhidrosis (PHH), level of sympathectomy and CS, we performed the Chi-Square and Fisher Exact test. There were no significant associations between both variables and CS (Table 7).\n\nχ2 = Chi-square value, df=degree of freedom, aFisher exact test, Significant at p<0.05\n\nIn univariate logistic regression analysis, potential predictors of CS with p values of less than 0.25 were selected for entry into the multivariate analysis after ruling out multicollinearity and interaction. These independent variables were medical issues (Yes/No) (p=0.219), level of sympathectomy (T2-T3, and T2-T4) (p=0.044), and frequency of follow-up (one month and more than one month) (p=0.000). In the multivariate binary logistic regression analyses, a preliminary final model was determined using the stepwise method and contains only two independent variables: level of sympathectomy (p=0.020) and frequency of follow-up (p=0.000). The full model containing both predictors was statistically significant, χ2 (2, N=118) = 31.21, p<0.001, indicating that the model was able to distinguish between subjects who reported and did not report CS. The model as a whole explained between 23.8% (Cox and Snell R square) and 32.0% (Nagelkerke R square) of the variance in CS and correctly classified 69.6% of cases. Model fit assessment using the Hosmer-Lemeshow test indicated that the model was a good model fit to the data (p=0.935). The classification table indicated that the model has attained a good fit to the data (70% of subjects were correctly classified by the model). The AUC of the ROC curve was 0.771 (95%CI 0.686-0.855), which is higher than the cut-off value of 0.7, and indicated that the model could adequately discriminate between those subjects reporting or not reporting CS. The final model is presented in Table 8. Subjects who had undergone ETS at the level of T2-T3 were almost three times more likely than those who had ETS at the level of T2-T4 to report CS (adjusted OR=2.869, 95%CI 1.177-6.991, p=0.020). Subjects who had more than one follow-up after ETS were more than thirteen times likely than those having only one follow-up to report CS (adjusted OR=13.558, 95%CI 4.174-44.038, p<0.001).\n\ndf=degree of freedom, Wald= Wald Chi Squared value, B=Beta value\n\na. Variable(s) entered on step 1: Sympathectomy level, Follow-up\n\nCox & Snell R Square=0.238; Nagelkerke R Square=0.320\n\nClassification Table: Percentage correctly predicting CS=69.6%\n\n\nDiscussion\n\nThe success rate of ETS for primary hyperhidrosis and the rate of CS are often used to measure how effective ETS is for primary hyperhidrosis in a particular center. Having a high rate of success and also a high rate of CS decreases the overall satisfaction of the patients after surgery. That being said, CS which is severe in nature is the worse outcome we would expect post-ETS. Most patients reported to be happy with having mild CS compared to primary hyperhidrosis on their palm42.\n\nThe strength of our study focuses on the relatively large sample size with a good multiracial distribution among the major races in Malaysia compared to other studies internationally. It is also important to highlight that this is the first study of its kind in Malaysia that provides an excellent background of the success rate and occurrence rate of CS that could spearhead many future studies around primary hyperhidrosis and its treatment. But our study had slightly more males seeking ETS as a cure for primary hyperhidrosis compared to females (57% males versus 42% females). This differs from what many authors have reported as the majority of the papers1,2,5,43,44 found that the incidence of primary hyperhidrosis in men and women are the same. Most of our patients who had ETS were Malays (79.7%) which tallies with our ethic predominance in Malaysia, as Malay race is the majority in Malaysia. Our analysis also shows that age, gender or BMI did not affect the rate of CS in these patients. A well-known study investigating factors affecting the outcome following ETS by Jaffer et al.45 supports this notion as their findings also revealed that age, sex, and BMI did not affect the outcome of the surgery or the rate of CS.\n\nIn our study, the level of sympathectomy that was mainly used in this sample was T2-T3 that accounted for 55.9% of the surgeries followed by the T2, T3, and T4 level, which accounted for 41.5% of the surgeries. There were very little variations to the technique of sympathectomy between these two groups as both groups had their surgeries in a single center. Earlier in 1994, Herbst et al.46 described the CS rate of 67.4% in T2-T4 resection at his center. However, Scognamillo et al.47 reported that there were no differences between T2-T4 and T3-T4 sympathectomy in terms of efficacy and CS rates, the sample size for the T3-T4 group in our study was too small to be used as comparison for other two large groups. We had only three patients who underwent sympathectomy at levels T3-T4.\n\nThe mean duration of surgery in our center was 46.6 ±14.29 minutes. The duration of surgery varies in many studies but Ong et al.48 from our neighboring country, have reported that their mean duration of surgery was 42 minutes. The duration of surgery has dramatically reduced from the time ETS was introduced in 1930s. Impressively, the mean duration of stay in the hospital in our sample was only 3.5 ± 1.05 days. The data was skewed slightly by nine patients who had long duration of hospital stay due to postoperative complications.\n\nIn our analysis, 98.3% of patients had complete relief of palmar hyperhidrosis post-ETS. The reported success rates for ETS in curing palmar hyperhidrosis around the world ranges between 94 to 100% with minimal recurrence rates37,49–51. The degree of symptom resolution varied significantly according to regions affected by hyperhidrosis. The main indication for ETS in our group of patients was palmar hyperhidrosis. We noted that 42 out of 54 patients who also had axillary hyperhidrosis had partial reduction (60–90%) in their symptoms. Besides that, 74 out of 100 patients who also had plantar hyperhidrosis on top of their palmar hyperhidrosis had partial reduction (60–90%) in their symptoms as well. The success rates in treating axillary and plantar hyperhidrosis were reported to be lower, with higher failure rates compared to having ETS performed to treat palmar hyperhidrosis46,52. We can conclude that ETS does not only cure palmar hyperhidrosis, but also reduces plantar and axillary hyperhidrosis. Although techniques and methods used to interrupt these chains vary between studies, if the correct level is interrupted, the results can be mimicked and this is indicated from the excellent success rate in our center.\n\nThe primary aim of this procedure was to improve the patients’ quality of life; hence, the complications should be minimal and essentially reduced. As expected, the most common post-operative complication in our study was CS. In our center, the CS rate is lower than average reported rate, 58.5%. Out of the 58.5% patient who had CS, the majority of them (30.5%) had mild form of CS. Most of our patients considered CS as a minor drawback, which is much more tolerable compared to their former condition. Only eleven patients (9.3%) who had the severe form CS regretted having the surgery. Twenty-two patients (18.6%) developed moderate CS in our study; but we were unable to assess how it affects them compared to their initial condition. It would be interesting to have longer follow-up to find out the progression of CS and also quality of life. Most of our patient had single follow-up, hence, we were unable to comment on the follow-up progression of CS. It is a common assumption that CS vanishes over time, but based on recent literature, this assertion is probably wrong. Herbst et al.46 discovered that after 14 years of follow up, 67% of their patients still had CS. A 16-year follow-up in 2013 by Askari and colleagues53 revealed that 97.6% of the patients from the study still had CS.\n\nWe would also like to believe our CS rates would be lower if not confounded by our tropical climate in Malaysia, where the temperature is high throughout the year. This would lead to greater sweating, exaggerating the incidence of CS in patients. A paper published by Li et al.54 concluded that climate plays a significant role in incidences of CS. They looked at patients who are from a hotter climate such as Taiwan and the reported CS rates are as high as 98%. Gassot et al.23 also quoted this phenomenon in their paper. Among the areas affected by CS, the back was affected the most in our sample size. CS occurring at the back totaled to 29% out of 58.5% patient who had CS in our center (29%, n= 20). Back of thighs and back involvement was the second highest reported site for CS in our patients (18.8%, n=13) followed by the abdomen (13%, n=9). In our study, we can conclude that the back and trunk were the most common regions being affected by CS. A few authors34,52 have described that the most common affected region by CS was the posterior aspect of the trunk.\n\nApart from CS, 10.1% of patients from our center developed other post-operative complications. Seven patients or 5.9% of patients developed pneumothorax postoperatively in our study. Its incidences have been reported to be around 1% to 6% in most reports from the literature1. Four patients (3.4%) had severe pain requiring additional analgesia post operatively. One patient developed post sympathetic neuralgia and is still on follow up regularly. One case had permanent bradycardia post ETS which is said to be a rare complication of ETS. Its occurrence has been reported in the literature46,55 and certain patients may also need pacemakers if their resting heartbeat goes below 50 beats per minute.\n\nHaving said this, it has to be stressed again that it was still CS that has the greatest impact on the quality of life after ETS. Although the pathophysiology of CS remains unknown, surgeons have been trying to reduce the incidence by lowering the level of sympathectomy31,56. Other surgeons have suggested that lowering the level of sympathectomy could reduce severe CS57,58. A recent meta-analysis conducted by Cai SW et al.59, who claimed to be the first group that conducted a meta-analysis of randomized controlled trials (RCT) on ETS to assess the effect of whether lowering the level of sympathectomy could really reduce CS, confirmed the fact that lowering the level of sympathectomy could reduce CS and severe CS after ETS.\n\nOur study has also showed a similar finding that basically lowering the level of sympathectomy, T2-T4, reduces the incidence of CS significantly. This has been the main predictor in our study. We also noticed that those who came for follow-up for more than once were the ones with CS. It is likely that the persistence of CS troubled the patients leading to the further follow-ups, though further study to look at the impact of ETS on the quality of life of the patients is required.\n\nThis present study has two limitations. First, it was a retrospective cohort study based on a proforma (Supplementary File 1) rather than a tele-interview or medical evaluation. Being an observational study, temporal relationship was unable to be established for a few variables, especially the follow-up data. There is also lack of clarity in some of the follow-up notes, which could have aided the study in providing more information regarding the nature of the CS. Nevertheless, this type of data collection has the advantage that it permits patients to provide self-reported data in a setting that is free from medical influence. Certain data regarding the period that the patient noticed the cessation of palmar hyperhidrosis, onset of CS, progression of CS; and triggers to CS were not available or poorly documented.\n\nSecondly, the direct involvement of the investigator in gathering data could entail the possibility of observer bias. Measures to reduce bias were taken beforehand in order to negate the influence of this bias, specifically the project leader was not involved in data collection. Another limitation would be the difficulty to objectively measure the outcome. Responses gathered from the patient during follow-up are subjective in nature and solely depended on the description of the patient and the surgeon’s perception of these descriptions.\n\nMoving forward, to have a more substantiated and quality data regarding the success rate and incidence of CS and their locations, a larger prospective cohort study with a long-term follow-up in multiple centers would be ideal. A thorough criterion of describing CS or resolution of symptoms should be set universally among all centers to allow for more unified and harmonious data comparison. This would eliminate misreporting and decreasing the wide gap in documenting the incidences of CS. Patients should be followed up at least for five years to gather data on the recurrence of hyperhidrosis after CS in our climate.\n\nThere should be an increase in awareness among healthcare providers regarding primary hyperhidrosis and ETS as the most important curative method for it. The high success rate and low incidence of CS should be taken into account and be offered to patients who are suffering from this dreadful condition. The local health ministry should modify the management guidelines and protocols of primary hyperhidrosis to reflect the findings of this study as this the first ever study to report the success rate of ETS for primary hyperhidrosis and incidence of CS in Malaysia.\n\n\nConclusion\n\nWe conclude that ETS is a safe, effective and aesthetically remarkable procedure for the treatment of primary hyperhidrosis considering the high success rate with only half of the patients developing CS. Our results compare favourably with or exceed those other studies in terms of success rates and incidences of CS. The clinical implication of this study provides solid evidence to patients or healthcare practitioners by showing excellent safety profile, high success rate and low rates of CS occurrence in patients who had ETS done for primary hyperhidrosis. Taking into account the multiracial population and our climate, we provided answers to patients who were hesitant to undergo ETS at our local setting. We also hope that we managed to inspire researchers to look into this subject of primary hyperhidrosis and ETS as this condition has often been neglected in our society despite the profound impact it has on the daily lives of patients. This study, we believe, has provided the groundwork for higher quality studies in this field especially in this country.\n\n\nData availability\n\nDataset 1: Endoscopic thoracic sympathectomy data 10.5256/f1000research.14777.d20359860\n\nDataset 2: Endoscopic thoracic sympathectomy statistical analysis output file 10.5256/f1000research.14777.d20359961", "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 – Proforma used for data collection.\n\nClick here to access the data.\n\n\nReferences\n\nCerfolio RJ, De Campos JR, Bryant AS, et al.: The Society of Thoracic Surgeons expert consensus for the surgical treatment of hyperhidrosis. Ann Thorac Surg. 2011; 91(5): 1642–8. PubMed Abstract \| Publisher Full Text\n\nHashmonai M, Kopelman D, Assalia A: The treatment of primary palmar hyperhidrosis: a review. 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Ann Thorac Surg. 2008; 85(5): 1747–51. PubMed Abstract \| Publisher Full Text\n\nLai CL, Chen WJ, Liu YB, et al.: Bradycardia and permanent pacing after bilateral thoracoscopic T2-sympathectomy for primary hyperhidrosis. Pacing Clin Electrophysiol. 2001; 24(4 Pt 1): 524–5. PubMed Abstract \| Publisher Full Text\n\nLin CC, Telaranta T: Lin-Telaranta classification: the importance of different procedures for different indications in sympathetic surgery. Ann Chir Gynaecol. 2001; 90(3): 161–6. PubMed Abstract\n\nLicht PB, Pilegaard HK: Severity of compensatory sweating after thoracoscopic sympathectomy. Ann Thorac Surg. 2004; 78(2): 427–31. PubMed Abstract \| Publisher Full Text\n\nYazbek G, Wolosker N, de Compos JR, et al.: Palmar hyperhidrosis--which is the best level of denervation using video-assisted thoracoscopic sympathectomy: T2 or T3 ganglion? J Vasc Surg. 2005; 42(2): 281–5. PubMed Abstract \| Publisher Full Text\n\nCai SW, Shen N, Li DX, et al.: Compensatory sweating after restricting or lowering the level of sympathectomy: a systematic review and meta-analysis. Clinics (Sao Paulo). 2015; 70(3): 214–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMusa AF, Gandhi VP, Dillon J, et al.: Dataset 1 in: A retrospective review on minimally invasive technique via endoscopic thoracic sympathectomy (ETS) in the treatment of severe primary hyperhidrosis: Experiences from the National Heart Institute, Malaysia. F1000Research. 2018. Data Source\n\nMusa AF, Gandhi VP, Dillon J, et al.: Dataset 2 in: A retrospective review on minimally invasive technique via endoscopic thoracic sympathectomy (ETS) in the treatment of severe primary hyperhidrosis: Experiences from the National Heart Institute, Malaysia. F1000Research. 2018. Data Source" }	[ { "id": "48307", "date": "28 May 2019", "name": "Nelson Wolosker", "expertise": [ "Reviewer Expertise Hyperhidrosis." ], "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 review of a well-studied subject: retrospective results of assisted video sympathectomy in a single center. In the Introduction the authors make a general review on the subject. There is a final paragraph in the Introduction defining the aims of the study, which are written within the Material and Methods section. Remove the aims from the Methods and put them in the last paragraph of the Introduction.\nThe casuistry is small, but significant. It is acceptable for a study demonstrating the results in Malaysia. The number of overweight and obese patients in this series is interesting. In international studies BMI greater than 25 generated greater intensity in SC.\nThe results are adequate, demonstrating that the sympathectomy in the Malaysian population is similar to international. The great variation of the levels of sympathectomy is also interesting, since articles from 2005 to 2007 already demonstrated advantages of sympathectomy at T3 or T4 levels alone over other levels of sympathectomy. But they reflect the local reality, which makes this data valid.\nThe conclusions are appropriate.\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": "65956", "date": "06 Jul 2020", "name": "Yongbing Chen", "expertise": [ "Reviewer Expertise thoracic 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 authors performed a retrospective study on the role of ETS in treating primary hyperhidrosis and found that ETS is a safe, effective and aesthetically remarkable procedure for the treatment of primary hyperhidrosis with only half of the patients developing mild to moderate degree of compensatory sweating. However, the findings are actually not novel enough except the predictors of CS. In addition, the number of patients was too small to provide robust evidence for practice. Meanwhile, ETS procedure with two incisions in their institution seemed outdated since uniportal ETS is routinely performed in our center. Generally, the study is well-designed and the statistical methods seem sound, but the findings are not very novel. Last but not least, some grammatical errors exist. These errors should be corrected and more limitations should be acknowledged before the manuscript is acceptable.\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?\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/7-670
https://f1000research.com/articles/7-666/v1	25 May 18	{ "type": "Research Article", "title": "Comparative study of the environmental impact of models of conventional agricultural and agro-ecological agriculture in the agricultural phase of tomato cultivation", "authors": [ "Carina Maribel Taipe Velasco", "Ronnie Xavier Lizano Acevedo", "Bence Mátyás", "Carina Maribel Taipe Velasco", "Bence Mátyás" ], "abstract": "Background: In this study, the water footprint and the carbon footprint were calculated during the activities of the agricultural phase of tomato cultivation, comparing agro-ecological production systems with conventional production systems. Methods: We examined with six plots in total: 3 agro-ecological plots and 3 conventional plots in la Esperanza and Tabacundo, Pedro Moncayo canton, Ecuador. The water footprint was calculated according to Hoekstra’s method. For the greenhouse gas emissions calculation, due to the production of fertilisers, the activity data was multiplied by the emission factor. Phytosanitary emissions were calculated using the factor given by BioGrace. Results: For the conventional system the most representative footprint is that of blue water with 44.19 litres of water/kg of tomatoes, followed by the green water footprint with 14.42 litres of water/kg of tomato whilst the lowest value is 0.96 litres of water/kg of tomatoes for the grey water footprint. Conclusions: The results obtained show that an agro-ecological system is the most efficient in terms of consumption of resources. Its produce also have an added value for promoting sustainability, responsible consumption and a healthier diet. The generation of eco-labels can encourage the consumption of these by expanding markets for this production system.", "keywords": [ "life cycle assesment", "food systems", "agriculture", "environmental impacts" ], "content": "Introduction\n\nGreenhouse gas emissions from agriculture continue to rise on a global scale, although not as fast as emissions from other human activities. Having better information at a national level on emissions from agriculture1,2, live-stock, fisheries and forestry can help countries identify opportunities to reduce them, whilst pursuing objectives of food security, resilience and rural development and gaining access to global financing for implementation3. The agricultural sector is the sector that uses the most water, globally representing around 69% of all extraction, with household consumption at approximately 10% and industry at 21%4 The water footprint of a product is the total amount of water used to produce the goods or the service we use. In this study we calculated the green, blue and grey water footprint. The green water footprint is the amount of water in the root zone of the soil and evaporated, transpired or incorporated by plants. The blue water footprint is the amount of water that comes from surface or groundwater and is evaporated, incorporated into a product or taken from one body of water (for example irrigated agriculture have a blue water footprint). The grey water footprint \"is the amount of fresh water required to assimilate pollutants to meet specific water quality standards\" (see Water Footprint Network website). The agro-ecological agriculture is farming that “centers on food production that makes the best use of nature’s goods and services while not damaging these resources” (see More and Better report on a viable food future). It links ecology, culture, economics and society to create healthy environments, food production and communities in order to maintain the sustainable development (see Groundswell international page on agroecological farming). While the conventional agriculture also known as industrial agriculture, \"refers to farming systems which include the use of synthetic chemical fertilizers, pesticides, herbicides and other continual inputs, genetically modified organisms, concentrated animal feeding operations, heavy irrigation, intensive tillage, or concentrated monoculture production. Thus conventional agriculture is typically highly resource and energy intensive, but also highly productive\" see USDA factsheet on conventional farming In this study, emissions and water requirements for tomato cultivation in conventional production systems and agro-ecological production systems were calculated in the La Esperanza and Tabacundo parishes, Pedro Moncayo canton.\n\n\nMethods\n\nThe present study examines the carbon and water footprint of product during the activities of the agricultural phase of tomato cultivation (between 2nd June and 5th of September 2017) in La Esperanza and Tabacundo, Pedro Moncayo canton, Ecuador. The average temperature was 20 °C, the humidity was 66% and the total precipitation was 320,8 mm in the examined period (https://en.climate-data.org/location/719640/). Three agro-ecologically managed plots (GPS decimal degrees: Plot 1: Latitude: -0.811193, Longitude: -78.6955; Plot 2: Latitude: -0.809214, Longitude: -78.6362; Plot 3: Latitude: -0.811429, Longitude: -78.6318) and three conventionally managed plots (GPS decimal degrees: Plot 4: Latitude: -0.809021, Longitude: -78.6273; Plot 5: Latitude: -0.805316, Longitude: -78.6114; Plot 6: Latitude: -0.805312, Longitude: -78.6114) were analyzed in this study. For information about the size of the experimental areas and the applied fertilizers please see Dataset 1. The water footprint was calculated as established by Hoekstra et al. (2011)5. For agro-ecological production systems, the green water footprint and blue water footprint were calculated. They lack a grey water footprint since they do not incorporate synthetic fertilisers, whereas for the conventional system the green water footprint, blue water footprint and the grey water footprint were calculated. Agro-ecological systems:\n\nWater footprint = Green Water Footprint + Blue Water Footprint(m3/ton)\n\nConventional case:\n\nWater Footprint = Green Water Footprint + Blue Water Footprint + Grey Water Footprint(m3/ton) (Hoekstraetal., 2011)\n\nFor the Carbon Footprint calculation, the equation given by greenhouse gases (GHG) Protocol, World Resources Institute and wbcsd (2011)6 was used:\n\nkgCO2eq = Activity DataEmission Factor x GWP\n\nFor the (GHG) calculation in the conventional plots, due to the use of fuels, the equation given by the IPCC7 belonging to the all-terrain category was used. This equation allows one to obtain CO2 emissions according to the type of fuel- be it diesel or petrol- as applicable for each case.\n\nEmission=∑jFueljEFj\n\nSource: IPCC (2006a)\n\nwhere:\n\nEmission: total emissions expressed in KgCO2eq\n\nFuel: fuel consumption TJ\n\nEF: Emission factor (KgCO2eq TJ)\n\nj: fuel type\n\nFor the greenhouse gas emissions calculation, due to the production of fertilisers, the activity data was multiplied by the emission factor. Phytosanitary emissions were calculated using the factor given by BioGrace8. Regarding greenhouse gases, due to direct emissions of N2O, the contributions of nitrogen in managed soils were taken into account and for the study the equation given by the IPCC9 was adapted so that for the case studies it was applied as follows.\n\nFor the conventional systems, the equation was reduced to:\n\nN20 − NNcontributions = (FSN + FCR) * EF1\n\nSource: IPCC (2006c)9\n\nAnd for the agro-ecological systems:\n\nN2O− NNcontributions = (FON + FCR) * EF1\n\nSource: IPCC (2006c)9\n\nThe calculation of indirect emissions of N2O for managed soils was carried out by means of adapting equation 11.9 of the manual (IPCC, 2006c)7 to the case study, thus the applied equation was: Conventional systems:\n\nN2O(ADT) − N = (FSN * FracGASF) * EF4\n\nSource: IPCC (2006c)9\n\nAgro-ecological systems:\n\nN2O(ADT) − N = (FON * FracGASM) * EF4\n\nSource: IPCC (2006c)9\n\nFor the calculation of greenhouse gas emissions due to the use of fertilisers, the results of direct and indirect emissions of N2O were taken into account. Regarding the emissions from applying phytosanitary products, this section was considered only in the conventional plots since they apply pesticides for the prevention of pests, such as fungicides and insecticides. To perform the calculation, the amount employed in kg/hectare (ha) and the emission factor given by BioGrace8 were taken into account.\n\n\nResults\n\nFigure 1 shows the water requirements for each plot. Regarding the conventional system, the highest values of green and blue water footprints corresponded to plot 5 with 34.42 and 87.54 litres of water/kg of tomatoes, re-spectively. For the agro-ecological systems, the plot with the highest green and blue water footprint was plot number 1 with 16.46 and 42.54 litres of water/kg of tomatoes, respectively. In relation to the grey water footprint, the highest value was found for plot 5 with 2.59 litres of water/kg of tomato.\n\nIn relation to the green water footprint, on average an agro-ecological system requires 9.07 litres of water/kg of tomatoes. For the blue water footprint, it requires 32.04 litres of water/kg of tomatoes (Figure 2). For the conventional system the most representative footprint is that of blue water with 44.19 litres of water/kg of tomatoes, followed by the green water footprint with 14.42 litres of water/kg of tomato whilst the lowest value is 0.96 litres of water/kg of tomatoes for the grey water footprint.\n\nThese results show that conventional cultivation consumes 18.45 litres more water for every 1 kg of tomatoes in the La Esperanza and Tabacundo parishes of the Pedro Moncayo canton (Figure 3).\n\nTable 1 shows that the highest generation of emissions was in plot 2 for the agro-ecological system and plot 4 for the conventional system The average emissions for the agro-ecological system in the study area was 757.83kg of CO2/ha whilst it was 830.96kg of CO2/ha for the conventional system. In the agro-ecological system, the greatest generation of emissions is due to the use of biofertilisers with an average of 426.68kg CO2/ha, whilst for the conventional system it is due to the use of fuel with a 552.32kg CO2/ha on average.\n\n\nDiscussion\n\nIn the La Esperanza and Tabacundo parishes an agro-ecological system requires less water compared to a conventional system, since the latter consumes 18.45 litres more water to produce a kilogram of tomatoes. In terms of emissions, the agro-ecological system obviously generates less, since it does not include the use of fuels because all the activities are carried out manually.\n\nIn relation to the Water Footprint, we used the values provided by Villavicencio et al.10 as a referential Water Footprint for fresh tomatoes (grown in the Coquimbo Region in the Choapa basin in Chile). They found a total water footprint of 84.2l/kg for the central region whilst we found a total water footprint of 59.57l/kg of tomatoes in the conventional system and 41.11l/kg in the agro-ecological system. Our values reflect the characteristics of the study area.\n\nAs a whole, an agro-ecological system emits on average of 757.83kg CO2eq/ha whilst a conventional system emits on average 830.96kg CO2eq/ha. In the latter system, the greatest generation of emissions corresponded to the use of fuel, with an average of 552.32kg CO2eq/ha whilst for the agro-ecological system the greatest generation of emissions corresponded to the use of biofertilisers with 426.68kgCO2eq/ha.\n\nThe results obtained show that an agro-ecological system is the most efficient in terms of consumption of resources. Its produce also have an added value for promoting sustainability, responsible consumption and a healthier diet. The generation of eco-labels can encourage the consumption of these by expanding markets for this production system.\n\n\nData availability\n\nDataset 1: Experimental setup. Area of the lands, total crop production (Kg), fertilizer application rate on total crop production, concentration of NPK in each solid fertilizer, mount of NPK (Kg), Liquid fertilizer application rate on the total crop production, Concentration of NPK in each liquid fertilizer, Amount of NPK in liquid fertilizer (Kg), GPS coordinates 10.5256/f1000research.14334.d20375011\n\nDataset 2: Calculated water footprint for each plot. Green, blue and grey water footprint in l (water)/kg(tomato). 10.5256/f1000research.14334.d20375112\n\nDataset 3: Calculated carbon footprint for each plots. Total GHG emission (kg CO2 eq): Fuel (CO2, N2O), fertiliser production (N, K2O, P2O) fertiliser use, photosanitary. 10.5256/f1000research.14334.d20375213", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe present research was supported by Universidad Politécnica Salesiana and Secretaría de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT) [PIC-16-BENS-005].\n\n\nReferences\n\nMátyás B, Chiluisa Andrade ME, Yandun Chida NC, et al.: Comparing organic versus conventional soil management on soil respiration [version 1; referees: 2 approved]. F1000Res. 2018; 7: 258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBautista G, Mátyás B, Carpio I, et al.: Unexpected results in Chernozem soil respiration while measuring the effect of a bio-fertilizer on soil microbial activity [version 2; referees: 2 approved]. F1000Res. 2017; 6: 1950. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEmisiones de gases de efecto invernadero de la agricultura, silvicultura y otros usos de la tierra America Latina y el Caribe. FAO. 2014. Reference Source\n\nFAO. (s.f): Agua y cultivos uso agrícola del agua. Reference Source\n\nHoekstra AY, Chapagain AK, Aldaya MM, et al.: The Water Footprint Assessment Manual: Setting the Global Standar. 2011. Reference Source\n\nGHG Protocol, World Resources Institute y wbcsd: Greenhouse Gas Protocol. Product Life Cycle Accounting and Reporting Standard. World Resources Institute. Washington DC, USA: Greenhouse Gas Protocol. 2011. Reference Source\n\nDavies W, Harnisch J, Lucon O, et al.: CAPÍTULO 3: Combustión Móvil. En Directrices del IPCC de 2006 para los inventarios nacionales de gases de efecto invernadero, Energía. Japón:IGES. 2006a; 2: 1–78. Reference Source\n\nBioGrace: BioGrace complete list of standar values version Public. 2011. Reference Source\n\nEggleston ES, Buendia L, Miwa K, et al.: CAPITULO 11: Emisiones de N2O de los suelos gestionados y emisiones de CO2 derivadas de la aplicación de cal y urea. Directrices del IPCC 2006 para los Inventarios Nacionales de Gases de Efecto Invernadero, Agricultura, Silvicultura y Otros Usos de la Tierra. Japón: IGES 2006c; 4: 1–56. Reference Source\n\nVillavicencio, Riquelme y Pérez: Huella hídrica en Tomate Fresco. En Osorio U., Alfonso (ed). Determinación de la huella del agua y estrategias de manejo de recursos hídricos. Serie Actas N° 50. Instituto de Investigaciones Agropecuarias, Centro Regional de Investigación Intihuasi, La Serena, Chile, 2013; 211.\n\nTaipe Velasco CM, Lizano Acevedo RX, Mátyás B: Dataset 1 in: Comparative study of the environmental impact of models of conventional agricultural and agro-ecological agriculture in the agricultural phase of tomato cultivation. F1000Research. 2018. Data Source\n\nTaipe Velasco CM, Lizano Acevedo RX, Mátyás B: Dataset 2 in: Comparative study of the environmental impact of models of conventional agricultural and agro-ecological agriculture in the agricultural phase of tomato cultivation. F1000Research. 2018. Data Source\n\nTaipe Velasco CM, Lizano Acevedo RX, Mátyás B: Dataset 3 in: Comparative study of the environmental impact of models of conventional agricultural and agro-ecological agriculture in the agricultural phase of tomato cultivation. F1000Research. 2018. Data Source" }	[ { "id": "36277", "date": "27 Jul 2018", "name": "María Fernanda Cárdenas", "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 abstract, the presented results refer only to the water footprint calculated for the conventional system. Without the missing information, is not possible to follow your conclusions, presented in the abstract but not in the document.\n\nThe introduction of the manuscript does not provide the reader a context to understand the importance of this study. This is, for example, the relevance of agroecology, the current politics about it, or the significance of tomatoes in particular or agriculture in general for the region where the study was carried out. I believe the earlier would enhance the study.\n\nThe study sites and their location would be clearer presented in a table or in a figure. In a table would be interesting add, besides the coordinates, some information about the field characteristics, since I suspect it has a relationship with the results obtained. Particularly, it would be interesting if helps to explain the huge differences you found among plots.\n\nAlso, and related to the earlier, it would be necessary to justify why you decided to work with 3 plots in each assessed system. This is a big concern to me due to the differences presented in fig. 1... there is no explanation in the document and is it must be statistically supported.\n\nFig 2. and Fig. 3 are not relevant since they are not showing new information.\n\nFinally, it is important to review the references. I.e, #9 says IPCCc, but it actually refers to Eggleston et al. Also, the letter c does not have sense if there is no a or b.\n\nIn general, English needs to be reviewed.\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": "36276", "date": "28 Aug 2018", "name": "Conrado Tobon", "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\nTitle and introduction The title of the manuscript does not provide a precise summary of the paper's content, which is, it does not condense the paper's content, since this refers to environmental impacts of two types of land use, while the content is totally related to water footprint.\nI suggest to change the title for a better descriptive name, although simple and brief, but including both: Carbon and water footprint.\n\nMethods. The authors failed to present a comprehensive methodology to calculate the components of total footprints: Green, blue and grey. A full description of the methods used to calculate these specific footprints is required in order to understand results, and whenever necessary, compare results. Moreover, they should explain why is not necessary to calculate Grey water footprint, for this specific case.\nAuthors should complete the methodology.\n\nFigures. Figures 1, 2, and 3 should be more clear, and do not requires number on the bars. That´s why the Y axis exists.\n\nDiscussion In the first paragraph of the discussion the authors again present the results, which in fact belong more to the results than to properly to the discussion component. As for the other paragraphs, the presented discussion is far too poor, and do not interpret and describe the significance of the findings in light of what was already known in this field of the science. It needs to be completed.\nThe authors should present a new discussion based on:\nAuthors should demonstrate their ability to think critically about the reported and formulate through the discussion session a more profound understanding of the research problem under investigation,\n\nTo denote possible implications of the results and explore possible improvements that can be made in order to further develop the research topic\n\nTo state how the findings from this study revealed new gaps in the literature that had not been previously exposed or adequately described.\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?\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/7-666
https://f1000research.com/articles/6-1706/v1	19 Sep 17	{ "type": "Research Article", "title": "Effectiveness of model-based clustering in analyzing Plasmodium falciparum RNA-seq time-course data", "authors": [ "Jelili Oyelade", "Itunuoluwa Isewon", "Damilare Olaniyan", "Solomon O Rotimi", "Jumoke Soyemi", "Itunuoluwa Isewon", "Damilare Olaniyan", "Solomon O Rotimi", "Jumoke Soyemi" ], "abstract": "Background: The genomics and microarray technology played tremendous roles in the amount of biologically useful information on gene expression of thousands of genes to be simultaneously observed. This required various computational methods of analyzing these amounts of data in order to discover information about gene function and regulatory mechanisms. Methods: In this research, we investigated the usefulness of hidden markov models (HMM) as a method of clustering Plasmodium falciparum genes that show similar expression patterns. The Baum-Welch algorithm was used to train the dataset to determine the maximum likelihood estimate of the Model parameters. Cluster validation was conducted by performing a likelihood ratio test. Results: The fitted HMM was able to identify 3 clusters from the dataset and sixteen functional enrichment in the cluster set were found. This method efficiently clustered the genes based on their expression pattern while identifying erythrocyte membrane protein 1 as a prominent and diverse protein in P. falciparum. Conclusion: The ability of HMM to identify 3 clusters with sixteen functional enrichment from the 2000 genes makes this a useful method in functional cluster analysis for P. falciparum", "keywords": [ "Genomics", "Plasmodium falciparum", "microarray", "Hidden markov model", "clustering", "Functional Enrichment." ], "content": "Introduction\n\nTechnological advancement in bioinformatics such as the high through put sequencing technology has resulted in the availability of a very large amount of informative data1. Expressions of thousands of genes are now being measured concurrently under various experimental conditions using microarray technology. Microarray consists of many thousands of short, single stranded sequences, each immobilized as individual elements on a solid support, that are complementary to the cDNA strand representing a single gene. Gene expression measurements can be obtained for thousands of genes simultaneously using microarray technology. In a cell, genes are transcribed into mRNA molecules which in turn can be translated into proteins, and it is these proteins that perform biological functions, give cellular structure and in turn regulate gene expression2.\n\nVarious researches had been carried out on the analysis and extraction of useful biological information such as detection of differential expression, clustering and predicting sample characteristics3. One of the important of gene expression data is the ability to infer biological function from genes with similar expression patterns4. Due to large number of genes and complexity of the data and networks, research has suggested clustering to be a very useful and appropriate technique for the analysis of gene expression data which can be used to determine gene coregulation5, subpopulation6, cellular processes, gene function7 and understanding disease processes8.\n\nClustering algorithms as described by9 can either be distance based or model based. Distance-based clustering such as the k means10 does not consider dependencies between time points while the model based approach embeds time dependencies and uses statistical models to cluster data. Other approaches include Self Organizing Maps11, Principal Component Analysis, hierarchical clustering4, graph theory approach12, genetic algorithms and the Support Vector Machine13. These algorithms has been successfully applied to various time series data but still have various shortcoming such as determining the optimal number of clusters and choosing the best algorithm for clustering since most of them are based on heuristics. The algorithms are also not very effective especially when a particular gene is associated with different clusters and performs multiple functions.\n\nThis research focuses on clustering of genes with similar expression patterns using Hidden Markov Models (HMM) for time course data because they are able to model the temporal and stochastic nature of the data. A Markov process is a stochastic process such that the state at every time belongs to a finite set, the evolution occurs in a discrete time and the probability distribution of a state at a given time is explicitly dependent only on the last state and not on all the others. This refers to as first-order Markov process (Markov chain) for which the probability distribution of a state at a given time is explicitly dependent only on the previous state and not on all the others. That is, the probability of the next (“future”) state is directly dependent only on the present state and the preceding (“past”) states are irrelevant once the present state is given14. Fonzo et al.14 defined HMM as a generalization of a Markov chain in which each (“internal”) state is not directly observable (hidden) but produces (“emits”) an observable random output (“external”) state, also called “emission” state. Schliep et al.9 proposed a partially supervised clustering method to account for horizontal dependencies along the time axis and to cope with the missing values and noise in time course data. This approach used k means algorithm and the Baum welch algorithm for parameter estimation. Further analysis on the cluster was done with the Viterbi algorithm which gives a fine grain, homogeneous decomposition of the clusters. The partial supervised learning was done by adding labeled data to the initial collection of clusters. Ji et al.15 developed an application to cluster and mine useful biological information from gene expression data. The dataset was first normalized to a mean of zero and variance of one and then discretized into expression fluctuation symbol with each symbol representing either an increase, decrease or no change in the expression measurement. A simple HMM was constructed for these fluctuation sequences. The model was trained using the Baum-Welch EM algorithm and the probability of a sequence given a HMM was calculated using the forward-backward algorithm. Several copies of the HMM was made so that each copy represent a single cluster. These clusters were made to specialize by using a weighted Baum-Welch algorithm where the weight is proportional to the probability of the sequence given the model. The Rand Index and Figure of Merit was used to validate the optimal number of cluster results.\n\nGeng et al.16 developed a gene function prediction tool based on HMM where they studied yeast function classes who had sufficient number of open reading frame (ORF) in Munich Information Center for Protein Sequences (MIPS). Each class was labeled as a distinct HMM. The process was performed on three stages; the discretization, training and inference stages and data used for this analysis was the yeast time series expression data. Lees et al.17 proposed another methodology to cluster gene expression data using HMM and transcription factor information to remove noise and reduce bias clustering. A single HMM was designed for the entire data set to see if it would affect clustering results. Each path in the HMM represents a cluster, transition between states in a path is set to a probability of 1 and transition between states on different path is set to a probability of 0. Genes were allocated to clusters by calculating the probability of each sequence produced by the HMM. Clusters were validated using the likelihood ratio test which computes the difference in the log-likelihood of a complex model to that of a simpler model. Zeng and Garcia-Frias18 implemented the profile HMM as a self-organizing map, this profile HMM is a special case of the left to right inhomogeneous HMM which is able to model the temporal nature of the data. This makes it very useful for real life applications. The profile HMM is trained using the Baum-Welch algorithm19 and clustering was done using the Viterbi algorithm and the algorithm was implemented on the fibroblast and the sporulation datasets. Beal et al.20 implemented the Hierarchical Dirichlet Process Hidden Markov Model (HDP-HMM) for clustering gene expression data with countably infinite HMM. Gibbs sampling method was used to reduce the time complexity of the inference. The data used for the implementation was derived from Lyer et al.21 and Cho et al.22 which was normalized and standardized to a log ratio of 1 at time t = 1. Baum Welch algorithm was used for Estimation Maximization. In this work, we adopted the work of Lee et al.17 and applied HMM on the Plasmodium falciparum RNA-seq dataset.\n\n\nMaterials and methods\n\nIn this work, data was extracted and normalized to a mean of 1 and standard deviation of 0. Discretization was done on the data to improve the clustering results. The HMM forward-backward algorithm and Baum-Welch training algorithm was implemented to cluster the gene expression data. Genes were then assigned to cluster using the forward algorithm and inference was done by obtaining functionally enriched genes in the cluster set using FunRich tool. The data used was published by 23, they used the Illumina based sequencing technology to extract expressions of 5270 P. falciparum genes at seven different time points every 8hrs for 42hrs. The clustering algorithms was implemented by first randomly initializing all the HMM parameters, then, forward algorithm was implemented to calculate the forward probabilities of the observation and Baum-Welch algorithm was used for data fitting, then the likelihood of each HMM was calculated iteratively until the optimal likelihood is obtained. This process is repeated for all the different sized HMMs used.\n\nHMMs can be viewed as probabilistic functions of a Markov chain24 such that each state can produce emissions according to emission probabilities.\n\nDefinition 1. (Hidden Markov Model). Let O = (O1,…) be a sequence over an alphabet ∈. A Hidden Markov Model λ is determined by the following parameters:\n\n\n\n• Si, the states i = 1, … N\n\n• πi, the probability of starting in state Si,\n\n• αij, the transition probability from state Si to state Sj, and\n\n• bi(ω), the emission probability function of a symbol ω ∈ Σ in state Si.\n\nDefinition 2. (Hidden Markov Cluster Problem). Given a set O = {O1, O2,…, On} of n sequences, not necessarily of equal length, and a fixed integer K ≪ n. Compute a partition C = (C1, C2,…, Ck) of O and HMMs λ1,…, λk as to maximize the objective function\n\nWhere L(Oi \| λk) denotes the likelihood function for generating sequence Oi by model λk:\n\nThe preprocessing was done in two stages. The first stage was the normalization and the second stage was discretization. The normalization was done with the R statistical package using the normalize library. Normalization removes static variation in the microarray experiment which affects the gene expression level. Normalization also helps in speeding up the learning phase. Missing values are also removed during normalization. Discretization was done by converting the time points to symbols depending on whether the expression value has increased, decreased or not changed. This is done by using the equation below.\n\n\n\nWhere:\n\nSi = fluctuation level between time i and i + 1\n\nEi = expression level at time point i\n\nL = number of time points.\n\na = threshold value between timepoints.\n\nIn this work, we implemented a model-based HMM clustering algorithm where a cluster represents a path in the HMM model. Therefore, as the number of cluster increases, the number of paths through the model increases and the HMM becomes larger and larger. The number of hidden state is the number of clusters multiplied by the sequence length. Research has also shown that HMM that transverse from left to right best models time series data, therefore HMM moves from only right-to-left. The structure of the HMM is shown in Figure 1.\n\nThe number 0, 1, and 2 represents the emmission symbols at each state.\n\nThe left to right model has the property that, as the time increases, the state transition increases, that is, the states moves from left to right. This process is conveniently able to model data whose properties change over time.\n\nThe forward algorithm calculates the forward probabilities of a sequence of observation. This is the probability of getting a series of observation given that the HMM parameters (A, B, π) are known. This computation is usually expensive computationally. The time invariance of the probabilities can however be used to reduce the complexity of this algorithm by calculating the probabilities recursively. These probabilities are calculated by computing the partial probabilities for each state from times t = 1 to t = T. The sum of all the final probabilities for each states is the probability of observing the sequence given the HMM and this was used in this research to compute the likelihood of a sequence given the HMM. The algorithm is in 3 stages and illustrated as follows:\n\nInitialization stage\n\nThis initializes the forward probability, which is the joint probability of starting at state and initially observing O1.\n\n\n\nInduction stage\n\n\n\nThis is the joint probability of observation and state 3 at time t + 1 via state at time t (i.e. the joint probability of observing o at state 3 at time t+1 and form state and time t). This is performed at all states and is iterated for all times from t = 1 to T-1.\n\nTermination stage\n\nThis computes all the forward variables, which is the P(o\|M).\n\n\n\nWhere:\n\nαij = transition from state i to j\n\nbi(o) = probability of emitting a symbol in a particular state\n\nt = time\n\nM = model\n\nαi = forward variable of state i\n\nπ = probability of starting at a particular state\n\nThe backward algorithm like the forward algorithm calculates backward probabilities instead. The backward probability is the probability of starting in a state at a time t and generating the rest of the observation sequence Ot + 1,…, OT. The backward probability can be calculated by using a variant of the forward algorithm. Therefore, instead of starting at time t = 1, the algorithm starts at time t = T and moves backwards from OT to Ot + 1. In this work, the backward algorithm was used alongside the forward algorithm to re-estimate the HMM parameters. This algorithm also involves three steps and is illustrated below.\n\nInitialization\n\nThis is the initial probability of being in a state Si at time T and generating nothing. The value of this computation is usually 1.\n\n\n\nInduction\n\nThis step calculates the probabilities of partial sequence observation from t+1 to end given state at time t and the model λ.\n\n\n\nTermination\n\nIt calculates all the backward variables which is the P(Ot+1, …, OT\|Q1 = Si).\n\n\n\nWhere:\n\nαij = transition from state i to j\n\nbj(o) = probability of emitting a symbol in a particular\n\nβt(j) = backward variable of state i in time t\n\nThe Baum-Welch algorithm, sometimes called the forward-backward algorithm makes use of the results derived from this algorithm to make inference. The Baum-Welch algorithm is a special form of the Expectation Maximization algorithm used for finding the maximum likelihood estimate of the parameters of the HMM. It was used in this work to train the various sized HMM parameters.\n\nThe E part of the algorithm calculates the expectation count for both the state and observation. The expectation of state count is denoted by γt(i). It is the probability of being in state at time t giving observation sequence and the model.\n\n\n\nThe expectation of transition count is denoted by εij(t). it is the probability of being in state Si at time t and state Sj at time t + 1 given an observation sequence and the model.\n\n\n\nBased on the expectation probabilities, we can now estimate the parameters that will maximize the new model. This is the M step of the algorithm.\n\nThe new initial probability distribution can be calculated as follows.\n\n\n\nThe re-estimated transition probability distribution is also calculated as follows:\n\nFinally, the new observation matrix is calculated using the formula below\n\nThe pseudo code of the training algorithm is represented below:\n\ni. Begin with some randomly initialized or preselected model, π\n\nii. Run O through the current model to estimate the expectations of each model parameter using the forward backward algorithm.\n\niii. Change the model to maximize the values of each path using the new π′, α′ij and b′i(t).\n\niv. Repeat until change in log likelihood is less than a threshold value or when the maximum number of iterations is reached.\n\nFor global optimal results, this algorithm is usually iterated depending on the size of the dataset.\n\n\nResults and discussion\n\nAfter implementing the HMM algorithms, the discretized data was parsed into the program. The dataset was trained using HMMs with cluster size from cluster 2 to 10. The likelihood of each HMM was calculated using likelihood ratio test and three clusters were identified. The likelihood ratio test of the dataset from cluster 2 to 10 is shown in Table 1 below. A positive LRT show that increasing the number of parameters is still worthwhile while a negative LRT shows that increasing the parameter would not give a better model. From the calculations below, the optimal number of clusters in the dataset based on the likelihood ratio test is 3. The log likelihoods of each cluster is also illustrated in Figure 2 below. It shows that the log likelihood increases with more parameters added initially but from cluster 3, there was no significant increase in log likelihood showing that the optimal number of clusters has been attained.\n\nThe LRT becomes negative after three (3) clusters giving the optimal number as 3.\n\nThe log likelihood values increase with the number of clusters sizes. After 3 clusters, there is not a significant increase in the log likelihood.\n\nThe dataset was trained using the Baum-Welch algorithm and the probability that an observation sequence belongs to a cluster was calculated using the forward algorithm. Genes with discretized value of zeros were also removed. After the likelihood ratio test was calculated and the optimal number of clusters found, each data was then separated into clusters using the forward algorithm. The first cluster consists of 502 genes, the second cluster had 481 genes and the third cluster had 668 genes. These results are represented in the Figure 3.\n\nThe x-axis shows the likelihood of each genes in each of the cluster and the y-axis shows the total number of genes in each cluster.\n\nErythrocyte membrane protein1(pfEMP1) –Clusters 1 and 2\n\nThe clustering algorithm efficiently clustered the differentially expressed genes into 3 clusters based on their functions. It is noteworthy that the most abundant genes are the erythrocyte membrane protein 1 (PfEMP1). The proteins are grouped in cluster 1 and cluster 2. PfEMP1 is an ubiquitously expressed protein during the intraerythrocytic stage of the parasite growth that determined the pathogenicity of P. Falciparum25. The virulence of P. falciparum infections is associated with the type of P. falciparum PfEMP1 expressed on the surface of infected erythrocytes to anchor these to the vascular lining. PfEMP1 represents an immunogenic and diverse group of protein family that mediate adhesion through specific binding to host receptors25. The Var genes encode the PfEMP1 family, and each parasite genome contains ~60 diverse Var genes. The differential expression of the proteins in this family has been reported to determine morbidity from P. falciparum infection26. This differential expression during infection and among patients could have accounted for its clustering into 2 different clusters by the algorithm.\n\nApart from clustering cell adhesion proteins into a sub-cluster, the algorithm also clustered the proteins involved in actin binding, transmembrane transportation and ATP binding. While the genes involved in ATP binding a largely conserved with their functions yet to be experimentally determined, the transport proteins are bet3 transport protein and aquaporin. Aquaporin is a membrane spanning transport proteins that is essential in the maintenance of fluid homeostasis and transport of water molecules and it has been identified as good therapeutic target27. Bet3 transport protein on the other hand is involved in the transport of proteins by the fusion of endoplasmic reticulum to Golgi transport vesicles with their acceptor compartment28.\n\nThe second cluster also has sub-cluster of PfEMP1 with other sub-clusters for GTP and ATP binding /ATP-dependent helicase activity as well as structural components of ribosome and ubiquitin protein ligase activity. Apart from PfEMP1, other sub-clusters are involved in protein turnover-protein synthesis and degradation29. These include the RNA helicases that prepare the RNA for translation and initiate translation, the ribosomal and GTP binding proteins that are integral part of the ribosome assembly involved in translation and the ubiquitin-conjugating enzymes are carry out the ubiquitination reaction that targets a protein for degradation via the proteasome30–32.\n\nThe genes coding for proteins involved in catabolic activities such as breakdown of proteins and hydrolysis of lipids are clustered in cluster 3. This cluster also included sub-clusters for genes involved in motor activity and DNA structural elements. The DNA structural elements include histone proteins and transcriptional initiator elements which are involved in epigenetic control of gene expression33. The ability of P. falciparum to grow and multiply both in the warm-blooded humans and cold-blooded insects is known to be under tight epigenetic regulation and it has been suggested as a good therapeutic target25.\n\nThe Functional Enrichment analysis tool was used to determine functionally enriched genes in each cluster. Each cluster was loaded separately based on their unique gene identifier. Genes are matched against the UniProt background database or by using a custom database that allows users load their own predefined Gene Ontology Term (GOTerm). We used the custom database and loaded annotations downloaded from PlasmoDB for our functional enrichment. The result of the 3 clusters is summarized in Table 2. FunRich was only able to identify 164 of the 502 genes in cluster 1. Cluster 1 has five functional annotations, 7.3% of the genes are functionally annotated with cell adhesion molecule, receptor activity, 1.2% are annotated with acting binding, 1.2% with transporter activity and 3.7% with ATP binding as shown in Figure 4. The remaining genes has no GO functions. We can deduce that since these genes are in the same cluster, they are likely to have functions as the genes with GO annotation.\n\nCluster 1 has four GO annotations, cluster 2 has five and cluster 3 has seven functional annotations.\n\nThe x-axis shows the percentage genes in the cluster with specific functional annotation while the y-axis shows the function of these genes.\n\nIn cluster 2, FunRich was able to identify 308 genes. In cluster 2, 7.1% of the genes are functionally annotated with the structural constituent of the ribosome, 3.9% with cell adhesion molecule receptor activity, 1.6% with ATP binding and ATP-dependent in a helicase activity, 1.0% with ubiquitin protein ligase activity and 1.3% with GTP binding which gives a total of five functional annotations as shown in Figure 5. The rest of the genes in cluster 2 are also predicted to have similar functions as with the genes with known GO annotation.\n\nThe x-axis shows the percentage genes in the cluster with specific functional annotation while the y-axis shows the function of these genes.\n\nIn cluster 3, FunRich was able to identify 249 of the 668 genes in the cluster set. This cluster has the largest GO annotation as it is enriched with seven functions. 2.8% of the genes are enriched with cysteine-type peptide activity, 2.4% with endopeptidase activity, 1.6% with hydrolase activity, 0.8% with ATP binding, action binding and monitor activity, 1.2% with DNA binding, 0.8% with unfolded protein binding and 0.8% with DNA binding, protein heterodimerization activity as shown in Figure 6. We can also deduce that in cluster 3, the genes with unknown functions are also predicted to have the same functions as with the known ones.\n\nThe x-axis shows the percentage genes in the cluster with specific functional annotation while the y-axis shows the function of these genes.\n\n\nConclusion\n\nClustering has been found to be a very useful technique in analyzing gene expression data. It has the ability to display large datasets in a more interpretable format. Several approaches have been developed to cluster gene expression data. The HMM has a better advantage over them because of its strong mathematical background and its ability to model gene expression data successfully. The HMM algorithms were implemented to perform cluster analysis on the P. falciparum gene expression dataset. 2000 genes were used and 3 clusters were identified. Sixteen major functional enrichment were identified for the clusters.\n\n\nData availability\n\nThe data sets used in this study are freely available in www.ncbi.nlm.nih.gov/pubmed/20141604", "appendix": "Competing interests\n\n\n\nThere is no competing interests declared.\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 Covenant University for providing the platform to carrying out this research.\n\n\nReferences\n\nHuang QW, Wu LY, Qu JB, et al.: Analyzing time-course gene expression data using profile-state hidden Markov model. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLavstsen T, Turner L, Saguti F, et al.: Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children. Proc Natl Acad Sci U S A. 2012; 109(26): E1791–E1800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeena LS, Rajni: Cloning and characterization of engA, a GTP-binding protein from Mycobacterium tuberculosis H37Rv. Biologicals. 2011; 39(2): 94–99. PubMed Abstract \| Publisher Full Text\n\nNandi D, Tahiliani P, Kumar A, et al.: The ubiquitin-proteasome system. J Biosci. 2006; 31(1): 137–155. PubMed Abstract\n\nRossi G, Kolstad K, Stone S, et al.: BET3 encodes a novel hydrophilic protein that acts in conjunction with yeast SNAREs. Mol Biol Cell. 1995; 6(12): 1769–1780. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRottmann M, Lavstsen T, Mugasa JP, et al.: Differential expression of var gene groups is associated with morbidity caused by Plasmodium falciparum infection in Tanzanian children. Infect Immun. 2006; 74(7): 3904–39. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "27477", "date": "01 Nov 2017", "name": "Hilary Ann Coller", "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 the application of an approach to model-based clustering of RNA seq time course data to data on the life cycle of Plasmodium falciparum. The clustering was performed with an iterative training based approach based on Hidden Markov Models. An advantage of this approach is that there is objective information on the optimal number of clusters that best fit the data, which was 3 in this case. The clusters that were discovered with this method separated proteins with distinct functional roles into different clusters. For instance, genes involved in catabolic activities were clustered together in cluster 3, which also contained some structural elements. The authors use software to clearly define the extent of enrichment of different functional categories in each of the three clusters identified. The findings may prove valuable to researchers working to identify new therapeutic targets to combat P. falciparum.\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": "28431", "date": "20 Dec 2017", "name": "Thomas D Otto", "expertise": [ "Reviewer Expertise Bioinformatics", "malaria", "genomics", "RNA-Seq" ], "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 paper describes an HMM clustering methods of a data set that I published.\nI would have liked to be able to run the model. It is difficult from the definitions to understand how it runs and performs. Therefore I would suggest that the author make the source code available. I will not further comment the cluster method itself.\nAssuming I would implement the method, I would not be able to replicate the analysis. The dataset has expression values from 7 time points: 0 hours - 48 hours for 5500 genes. They can be read counts or RPKM. It is not clear which input data were used and how those were then filtered ( alpha threshold in Le Roch et al.1).\nWe think that at least 3300 genes are expressed, but in this analysis, just 1700 are clustered. In the blood stage of plasmodium, expressed genes follow a clear pattern, as can be seen in Bozdech et al.2, and the results should be compared to this and other similar papers (at least mentioned in the introduction).\nTo emphasize the PfEMP1 is interesting, but those genes should not be expressed in parasites in culture(or at least their expression has no biological context), and they are expressed at around the same time.\nJust as a suggestion, the paper would also more robust if other datasets would be used and the results compared: López-Barragán et al.3\n\nMinor:\nThe dataset was generated from RNA-Seq data, not microarray, as mentioned in two parts of the paper.\n\nIt would be good to have the version of the GO terms. I think that those GO terms were significantly represented, what the cut-off?\n\n40% of Plasmodium genes are of unknown function. Those won't have any GO terms. I think is wrong to deduce that they are likely to have the same function if occurring in the same cluster.\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? 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? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "29434", "date": "31 Jan 2018", "name": "Ashis Das", "expertise": [ "Reviewer Expertise Malaria molecular biology", "transcriptomics" ], "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 discusses the application of HMM to RNA seq time course data from in-vitro P. falciparum culture to cluster the data into 3 clusters and 16 major functional enrichment was seen for these. Although there has been appreciable analysis performed there remains some major concerns. Some of the most important have been presented below for suitable incorporation.\n\nThe data pre-processing step mentioned in this article was not clear. What type of input data was used here and how the filtering was done? It was mentioned that normalize library from R package was used. Is it appropriate for normalizing RNA Seq based transcriptome studies, since it was mentioned as used for microarray studies (Is it upgraded for RNA Seq based studies?)\n\nWhat is the criterion used to assign the expression of a particular gene across seven different time points? It is unclear with the brief explanation of Discretization. Is this determining the expression status of a particular gene?\n\nAvailability of data used after normalization would help in reproducibility.\n\nOverall I suggest detailed explanation of methodology adopted and its execution with the data used. Proper discussion of the same is also necessary.\n\nThe RNA Seq based transcriptome data was generated from highly synchronous culture data. The discussion of PfEMP1 linking with the pathology is unnecessary and misleading. Especially the statement “This differential expression during infection and among patients could have accounted for its clustering into 2 different clusters by the algorithm” is not acceptable since the data is from in-vitro culture.\n\nI recommend the use of this method to other transcriptome based studies of P.falciparum at different time points (microarray, RNASeq) to validate the results obtained in the present study. Discussion about the results obtained in this study with other transcriptome based studies is also necessary. This will reveal the robustness of the approach employed in this paper.\n\nThe criteria for GO Term enriched (p-value) should be provided in the discussion although it has been mentioned only in the Figures.\n\nSince data was taken from different time points, the stochastic nature of the HMM process may not be appropriate to cluster gene expression data which have strong dependency.\n\nIt would be interesting to classify the var genes obtained in the different clusters. PfEMPI as encoded by the var gene family is divided into subfamilies with different functional domains. Are the different clusters showing segregation of different subfamilies? In the IDC time course experiment which was performed in-vitro based on which the RNA Seq data has been taken is there a difference in the var gene subfamily expression at different time points?\n\nAs mentioned in the materials and methods RNA seq based expression data of 5270 P.falciparum genes were considered. However in conclusion it has been mentioned that only 2000 genes were used for clustering. What is the implication of this statement?\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?\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": [ { "c_id": "3655", "date": "25 May 2018", "name": "Jelili Oyelade", "role": "Author Response", "response": "1. The data pre-processing step mentioned in this article was not clear. What type of input data was used here and how the filtering was done? It was mentioned that normalize library from R package was used. Is it appropriate for normalizing RNA Seq based transcriptome studies, since it was mentioned as used for microarray studies (Is it upgraded for RNA Seq based studies?) Response The input data was RNA-SEQ, The data was normalized to a mean of 1 and standard deviation of 0. Yes it is based on this paper. Zyprych-Walczak, J., Szabelska, A., Handschuh, L., Górczak, K., Klamecka, K., Figlerowicz, M., & Siatkowski, I. (2015). The impact of normalization methods on RNA-Seq data analysis. BioMed research international, 2015. 2. What is the criterion used to assign the expression of a particular gene across seven different time points? It is unclear with the brief explanation of Discretization. Is this determining the expression status of a particular gene? Response Discretization was conducted by converting the time points to symbols depending on whether the expression value has increased, decreased or not changed. This method has been used by Huimin Geng et al. (2003) and Lees et al (2007) and has been able to justify the reliability of this discretization process. 3. Availability of data used after normalization would help in reproducibility. Response The availability of the data used after normalization has been provided 4. Overall I suggest detailed explanation of methodology adopted and its execution with the data used. Proper discussion of the same is also necessary. Response The detailed explanation of the methodology has been improved and a proper discussion of the findings has been added 5. The RNA Seq based transcriptome data was generated from highly synchronous culture data. The discussion of PfEMP1 linking with the pathology is unnecessary and misleading. Especially the statement “This differential expression during infection and among patients could have accounted for its clustering into 2 different clusters by the algorithm” is not acceptable since the data is from in-vitro culture. Response “This differential expression during infection and among patients could have accounted for its clustering into 2 different clusters by the algorithm.” has been removed from discussion 6. I recommend the use of this method to other transcriptome based studies of P.falciparum at different time points (microarray, RNASeq) to validate the results obtained in the present study. Discussion about the results obtained in this study with other transcriptome based studies is also necessary. This will reveal the robustness of the approach employed in this paper. Response This is very good suggestion but, this work, is based on the work of Karen Lees , Jennifer Taylor ,Gerton Lunter , and Jotun Hein titled: “Identifying Gene Clusters and Regulatory Themes using Time Course Expression Data, Hidden Markov Models and Transcription Factor Information” which has actually proved the robustness of the approach. 7. The criteria for GO Term enriched (p-value) should be provided in the discussion although it has been mentioned only in the Figures. Response The GO criteria - “Statistical cut-off of enrichment analyses in FunRich software was kept as default with a p-value <0.05.” has been provided in the discussion. 8. Since data was taken from different time points, the stochastic nature of the HMM process may not be appropriate to cluster gene expression data which have strong dependency. Response Gene expression is a fundamentally stochastic process, with randomness in transcription and translation leading to significant cell-to-cell variations in mRNA and protein levels. Model-based clustering approaches are effective when structural properties of the data can be explicitly expressed and HMM can be used by these types of approaches to partition gene expression time-series data into clusters. The process starts with an initial collection of HMMs that encompass typical qualitative behavior and the process iteratively find cluster models such a way that the joint likely hood of the clustering is maximized. This method is also robust with respect to noisy and frequently missing data. 9. It would be interesting to classify the var genes obtained in the different clusters. PfEMPI as encoded by the var gene family is divided into subfamilies with different functional domains. Are the different clusters showing segregation of different subfamilies? In the IDC time course experiment which was perfomed in-vitro based on which the RNA Seq data has been taken, is there a difference in the var gene subfamily expression at different time points. Response Yes, it is possible, different clusters can also show segregation of different subfamilies and there is no difference in the var gene subfamily expression at different time points. 10. As mentioned in the materials and methods RNA seq based expression data of 5270 P.falciparum genes were considered. However in conclusion it has been mentioned that only 2000 genes were used for clustering. What is the implication of this statement? Response The normalization and discretization process reduced the data and we removed all missing values and noise reduce the data to about 2000 genes." } ] } ]	1	https://f1000research.com/articles/6-1706
https://f1000research.com/articles/7-662/v1	24 May 18	{ "type": "Research Article", "title": "Molecular characterization of hookworm spp. isolated from food handlers, Khartoum, Sudan: A cross-sectional study", "authors": [ "Tarig A. Gamar", "Hassan H. Musa", "Hisham N. Altayb", "Mohamed H. Mohamed", "Adam D. Abakar", "Hassan H. Musa", "Hisham N. Altayb", "Mohamed H. Mohamed", "Adam D. Abakar" ], "abstract": "Background: Hookworms infect the intestines, cause an itchy rash, respiratory and gastrointestinal problems, and eventually iron deficiency (anaemia) due to the ongoing loss of blood. The objectives of this study were to assess the prevalence and molecular characterization of hookworms isolated from food handlers attending the Public Health Laboratories in Khartoum state, Sudan, for annual check-ups, and to assess the efficiency of PCR as molecular probe for hookworm infection. Methods: A total of 350 foods handlers’ participant's stool samples who were not suspected to be infected with hookworms were studied. Conventional methods were applied to make an early diagnosis. Stool samples were collected from public health laboratories (the public health lab in the Medical Commission) of Khartoum State; Omdurman locality, Khartoum North locality and Khartoum locality between October 2016 and April 2017. Specific identification was made by PCR on specimens identified as positive by Baermann’s technique, which were then sequence and genotyped Results: The prevalence of hookworms in the stool samples of food-handlers was 1.43%. One larval specimen recovered by Baermann’s technique was confirmed to be Necator americanus by PCR. PCR also confirmed that Necator americanus was the common species isolated from four further specimens. The results of DNA sequencing for Necator americanus were deposited in NCBI GenBank under the following accession numbers: sample 91, MH035824; sample 92, MH035825; sample 294, MH035826; and sample 319 MH035827. Conclusion: PCR was found to be effective for confirmation of the diagnosis of hookworm infection and can aid the clinician in initiating prompt and appropriate antiparasite therapy.", "keywords": [ "Hookworm", "Genotyping", "Molecular sequencing", "Necator americanus", "Sudan" ], "content": "Introduction\n\nAncylostomatidae and strongyl nematodes were known to pose a burden among a variety of mammalian hosts, including humans1. Ancylostoma duodenale and Necator americanus are the species recorded as being most responsible for human infection. They are found in mostly in warm and tropical areas2, and infect around 1.3 billion people worldwide3. A. duodenale are located in Central, Eastern and Northern Africa, India, Australia and Europe4. Further, N. americanus is present in Sub-Saharan Africa, Eastern Asia and Southeast Asia4. A zoonotic species in Asia (A. ceylanicum), also causes human infection; however, this is of limited significance and its exact geological appropriation has not been depicted. The most widespread of all hookworms are A. caninum, a parasite that infects dogs, and has lately been affirmed to survive in the human gut (but without developing sexually)5. Laboratory diagnosis of hookworm infection, routinely based on the presence of eggs in faeces by direct microscopy and/or concentration techniques6. Sometimes after gathering, through faeces culture tests (after 24 hours), hookworm ova may have hatched and rhabditiform larvae might be visible; due to morphological similarity between the two, these larvae must be differentiated from Strongyloides larvae. The severity of disease can depend on the number of ova that are counted in faeces. Furthermore, in some cases, adult hookworms may be found6. Use of molecular techniques (such as PCR) for parasitic detection and identification is more accurate and effective than conventional methods, and requires DNA of the hookworms for detection. For example, identifying both types of internal transcript spacer (ITS1 and ITS2, individually) of ribosomal DNA (rDNA) are proven genetic markers of parasitic nematodes, including A. duodenale and N. americanus.\n\nThe aim of this study was to estimate the prevalence and molecular characterization of hookworms isolated from the stool of food handlers attending Public Health Laboratories in Khartoum state, Sudan, for annual check-ups.\n\n\nMethods\n\nA total of 350 stool samples were collected. A previously described formula was used to determine sample size7.\n\nFood handlers working in food facilities in Khartoum State, annually medically checked in public health laboratories and willing to participate were included in this study irrespective of their age, gender and nationality. The public health laboratories were located in Khartoum State, Omdurman locality, Khartoum North locality and Khartoum locality, with sample collection conducted between October 2016 and April 2017. The age of participant ranged from 16 to 68 years, with an average age of 32 years; 46% of the participants were less than 29 years old compared with 54% of being 29 or above. The majority of participants were males (83.19%), with 16.81% being female. Distribution of samples according to the residence data showed that 101 participants were from Khartoum north (28.9%), 160 participants were from Omdurman (45.7%) and 89 participants were from Khartoum (25.4%).\n\nIn the first stage, the collected specimens were examined using a microscope (Olympus CX22, Japan), the formol-ether concentration technique and Baermann’s technique, as described previously8. Positive detected samples (those that included hookworm ova/larvae) were examined using PCR and DNA sequencing techniques for genotyping. Fresh stool specimens were collected from public health labs in Khartoum State.\n\nSamples that were found to be positive for hookworms eggs by direct examination, or for larvae by Baermann’s technique, were selected for PCR testing (five samples). The stool samples that were negative for parasites by direct smear or the formol-ether concentration technique on three consecutive stool samples were used as negative controls. For molecular examinations, all stool samples were preserved in 70% ethanol at −20°C. The third-stage larvae that were recovered by Baermann’s technique (Filariform) were collected and preserved. The extracted DNA from filariform larvae were used as control DNA during the molecular assay using Biotechnology G-Spinтм Total DNA Extraction Kit (iNtRON Biotechnology, Inc.), according to the manufacturer’s protocol.\n\nDNA was extracted from stool as per manufacturer's instruction used iNtRON Biotechnology G-Spinтм Total DNA Extraction Kit (iNtRON Biotechnology, Inc.) according to the manufacturer’s instructions.\n\nOne primer pair was used: RTHW1F (forward): 5'-GAT GAG CAT TGC WTG AAT GCC G-3') and RTHW1R (reverse): 5'-GCA AGT RCC GTT CGA CAA ACA G-3'9.\n\nThe partial ITS1, full-length 5.8S gene, and partial ITS2 ribosomal DNA regions were amplified from larvae and ova using PCR. Amplicon sizes were approximately 485 bp (if it typical to N. americanus) or 380 bp (if it typical to Ancylostoma spp.). The procedure for single-round PCR amplification was performed according to Maxime PCR premix kit (iNtRON Biotechnology, Inc.) REF technique. Briefly 5 µl of DNA extract was added to PCR premix (Maxime PCR premix kit i-Taq), containing i-TagTM DNA polymerase, dNTP mixture and reaction buffer. Next, 2 µl primer (forward and reverse) was added alongside 13 µl of nuclease-free water. The reaction mixture was initially denatured at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 secs, annealing at 65°C for 30 secs and extension at 72°C for 30 sec. This was followed by a final extension step for 10 min at 72°C in a thermal cycler (SensoQuest GmbH, Germany).\n\nApproximately 5µl of PCR product was elecrophoresed on a 1.5% agarose gel (containing 1.5 µg/100 ml ethidium bromide) in Tris-borate-EDTA buffer, along with the tracking dye bromophenol blue, initially at 120 V and 35 A for 60 min. Thereafter, bands were visualized under UV light and an amplicon of 485 bp was considered positive for hookworm DNA.\n\nDNA sequencing was carried out to confirm identification of the pathogen. Owing to the limited amount of DNA generated from one sample, only four samples of PCR products (485 bp) were sequenced using Sanger sequencing (Macrogen, Inc., Korea). The DNA fragment was 485 bp (if from N. americanus) or 380 bp (if it typical of Ancylostoma spp) from an internal sequence of the amplicon of single-round PCR were obtained using the specific primers. Two sequence fragments were generated for five samples, which were edited manually to correct possible base errors using BIOEDIT 7.09. They were then subsequently joined to reconstruct a fragment of 485 bp or 380 bp spanning genes for hookworm spp.\n\nDNA sequences were compared with the NCBI database to check DNA sequencing quality and specificity using the nucleotide BLAST server. Sequences were submitted in sequence alignment form to Clustal W (online tool) for multiple sequence alignment.\n\nFirstly, before uploading the sequences to NCBI, we proofread the nucleotide chromatogram using Finch TV software version 1.4.0 to ensure that all ambiguous sites were correctly called and to determine the overall quality. Next, nucleotides sequences were searched for sequence similarity using nucleotide BLAST10 Highly similar sequences were retrieved from NCBI and subjected to multiple sequence alignment using BIOEDIT software11.\n\nSamples were collected from participants after provision of informed consent. Ethical approval was obtained from the National Committee for Research, Ministry of Health, Khartoum State.\n\n\nResults\n\nIn this study, the stool samples of 350 participants were investigated for hookworms; five samples were found to be positive (1.43%) using the formol-ether concentration technique. One sample was found to be positive using Baermann’s technique, which was used as the positive control. Five samples were found to positive by PCR, as shown in Figure 1.\n\nLane M, 100 bp marker; lane 1, postive control 485 bp; lanes 2–5, postive samples; lane 6, negtive control; lane 7, negative sample.\n\nFour hookworm samples (91, 92, 294 and 319) were sequenced by Macrogen, Inc., Korea and the sequences uploaded to NCBI Genbank (accession numbers: MH035824 (sample 91), MH035825 (sample 92), MH035826 (sample 294) and MH035827 (sample 319). Using nucleotide BLAST, the sequence of samples 91, 92, 294 and 319 were queried for alignment. Samples 91 and 92 showed 100% similarity with N. americanus isolated genes for 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA (Figure 2). Sample 294 showed 98% similarity to N. americanus isolated genes for 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA (Figure 3). Sample 319 showed 97% similarity with N. americanus isolated genes for 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA, (Figure 4).\n\nRed lines indicate high identity; sample 91 showed 100% identity to Necator americanus genes for 18S rRNA, ITS1, 5.8S rRNA and ITS2.\n\nRed lines indicate high identity. Sample 294 showed 98% identity to Necator americanus genes for 18S rRNA, ITS1,5.8S rRNA and ITS2.\n\nRed lines indicate high identity. Sample 319 showed 97% identity to Necator americanus genes for18S rRNA, ITS1,5.8S rRNA and ITS2.\n\n\nDiscussion\n\nThe present study indicates that the prevalence of hookworms in food-handlers who attended for annual check-ups in Khartoum State, Sudan was 1.43%. To the best of our knowledge, this is the first study to identify hookworm infection in Sudan using molecular techniques; it can therefore serve as a baseline for studies of hookworms in Sudan. Molecular techniques are more advantageous for hookworm identification as they are rapid and more sensitive. DNA was extracted from larva and ova using the iNtRON Biotechnology G-Spinтм Total DNA Extraction Kit. Partial ITS1, full-length 5.8S and partial ITS2 rDNA regions were amplified and then sequenced. Molecular analysis was used to confirm human infections with one species of human hookworms, namely, N. americanus. The nucleotide sequences were analyzed by nucleotide BLAST searching and DNA was aligned using Clustal W13. Amplicon sizes were approximately 485 bp (typical of N. americanus). Sequences showed extremely high similarities (97–100%) with hookworm sequences in the GenBank database. The 4 samples studied were N. americanus. They were similar to N. americanus LC036565.1 (Japan), KM891738.1 (China) and LC036563.1 (Southern Vietnam). All sequences obtained in this study were deposited in the GenBank database. The results also indicate that PCR may be considered as a sensitive method for confirming a diagnosis of hookworm infection, and can aid the clinician in initiating prompt and appropriate antiphrastic therapy.\n\nThe differences in hookworm species populations causing human disease in different areas may possibly be related to parasite manner, ethnicity, atmosphere, temperature, and ecological factors2,14. The finding of N. americanus in Sudan goes well with other finding reported from Peninsular Malaysia15 and Thailand9, where N. americanus was more common than A. ceylanicum. Their findings also supported this study that A. duodenale infection was not found.\n\n\nConclusion\n\nPCR is a valuable tool for the laboratory diagnosis of parasites. It was found to be effective, sensitive and confirmatory for the diagnosis of hookworm infection and can aid the clinician in initiating prompt and appropriate antiparasite therapy. We confirmed that the major hookworm species infecting humans in Sudan is N. americanus.\n\n\nData availability\n\nDataset 1. Complete positive/negative results for each technique used to identify parasites in every stool sample. DOI: 10.5256/f1000research.14683.d20417612.\n\nSequence of sample 91, Accession number MH035824: http://identifiers.org/ncbigi/GI:1356678983.\n\nSequence of sample 92, Accession number MH035825: http://identifiers.org/ncbigi/GI:1356678984.\n\nSequence of sample 294, Accession number MH035826: http://identifiers.org/ncbigi/GI:1356678985.\n\nSequence of sample 294, Accession number MH035827: http://identifiers.org/ncbigi/GI:1356678986.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors extend their heartfelt thanks to the participants and staff for their support.\n\n\nReferences\n\nAnderson RC: Nematode Parasites of Vertebrates: Their Development and Transmission. (Cabi). 2000. Publisher Full Text\n\nBeaver PC, Jung RC, Cupp EW, et al.: Clinical Parasitology. (Lea & Febiger). 1984. Reference Source\n\nCrompton DW: How much human helminthiasis is there in the world? J Parasitol. 1999; 85(3): 397–403. PubMed Abstract \| Publisher Full Text\n\nde Silva NR, Brooker S, Hotez PJ, et al.: Soil-transmitted helminth infections: updating the global picture. Trends Parasitol. 2003; 19(12): 547–51. PubMed Abstract \| Publisher Full Text\n\nProciv P, Croese J: Human enteric infection with Ancylostoma caninum: hookworms reappraised in the light of a \"new\" zoonosis. Acta Trop. 1996; 62(1): 23–44. PubMed Abstract \| Publisher Full Text\n\nJayaram Paniker CK: Textbook of Medical Parasitology. (Jaypee Brothers Medical Publishers (P) Ltd). 2007. Publisher Full Text\n\nSathian B, Sreedharan J, Baboo SN, et al.: Relevance of Sample Size Determination in Medical Research. Nepal J Epidemiol. 2010; 1(1): 4–10. Publisher Full Text\n\nGarcia LS, Bruckner DA: Diagnostic Medical Parasitology. (American Society for Microbiology (ASM)). 1997. Reference Source\n\nPhosuk I, Intapan PM, Thanchomnang T, et al.: Molecular detection of Ancylostoma duodenale, Ancylostoma ceylanicum, and Necator americanus in humans in northeastern and southern Thailand. Korean J Parasitol. 2013; 51(6): 747–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltschul SF, Madden TL, Schäffer AA, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHall TA: Bioedit: A User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/Nt. In Nucleic acids symposium series. ([London]: Information Retrieval Ltd., c1979-c2000) 1999; 95–98. Reference Source\n\nGamar TA, Musa HH, Altayb HN, et al.: Dataset 1 in: Molecular characterization of hookworm spp. isolated from food handlers, Khartoum, Sudan: A cross-sectional study. F1000Research. 2018. Data Source\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): 4673–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoagland KE, Schad GA: Necator Americanus and Ancylostoma Duodenale: Life History Parameters and Epidemiological Implications of Two Sympatric Hookworms of Humans. Exp Parasitol. 1978; 44(1): 36–49. PubMed Abstract \| Publisher Full Text\n\nNgui R, Ching LS, Kai TT, et al.: Molecular identification of human hookworm infections in economically disadvantaged communities in Peninsular Malaysia. Am J Trop Med Hyg. 2012; 86(5): 837–42. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "38900", "date": "03 Oct 2018", "name": "Luis Fernando Viana Furtado", "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 propose the analysis of the presence of hookworms in 350 samples of food handlers from Sudan. From the total of positive samples, the authors did a PCR with primers specific for hookworms, confirming the species found by sequencing. Although the work has relevance, some points can be improved.\nAbstract It is not necessary to mention the collection period of the samples in the abstract. In the results of the abstract, I suggest that the authors mention the absolute number of infected persons after the percentage: \"The prevalence of hookworms in the stool samples of food-handlers was 1.43% (5/350).\"\nIt is not necessary to specify the accession numbers of the GenBank sequences in the abstract.\nAt the conclusion of the abstract, the authors do not say anything about the percentage of parasitized individuals. What is the impact of this prevalence on food handlers? What are the risks to consumers?\nIntroduction The citation of reference 3 is outdated. The most current prevalence of hookworm infection can be seen in Pullan RL, Smith JL, Jasrasaria R, Brooker SJ 2014. Global numbers of infection and disease burden of soil transmitted helminth infections in 2010. Parasit Vectors 7:37.\nMethodology It is not clear to me whether all 350 samples were analyzed by formaldehyde-ether concentration technique and Baermann's technique. If all samples were analyzed by these two methods, this should also be clear in the abstract.\nThe concentrations of the PCR components and DNA must be provided. The authors only provide the volumes used, but not the concentration. For reproduction of the technique, other researchers need the same conditions.\nI did not understand why the authors mentioned that the \"amplicon of 485 bp was considered positive for hookworm DNA\". What if the amplicon had 380 bp (corresponding to the species Ancylostoma ssp.)?\nDiscussion The authors should be more cautious about discussing molecular results. Although it is true that PCR is a more sensitive technique than conventional coproparasitological methods, the results of the work do not allow to conclude this. The authors do not screen all samples comparing the different methods. In addition, the authors could discuss the possibility of cross-reaction. How specific are these primers so that they can amplify only the genetic material of hookworms and not that of other nematodes? How feasible is the use of PCR for diagnosis in Sudan? Although PCR is a more sensitive technique, the ideal for the reality of many localities is still the combination of different coproparasitological methods, which, although requiring expertise, are inexpensive techniques.\nSome molecular techniques such as RFLP-PCR and qPCR have already been standardized for the determination of hookworm species. The authors should discuss something about these other techniques.\nAlthough the prevalence of hookworm infection has been low, the authors discuss nothing about the impact of parasitosis on food handlers. There are many papers that discuss the risks of this infection. Some speculation can be made.\nConclusion To conclude that Necator americanus is the most prevalent hookworm in Sudan based only on these results is very strong. The analysis of only 350 samples, with the molecular confirmation of only four of these samples does not allow to conclude something so strong.\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?\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": "36850", "date": "15 Oct 2018", "name": "Nicole Gottdenker", "expertise": [ "Reviewer Expertise Disease ecology", "wildlife pathology", "theoretical disease ecology", "vector borne and zoonotic diseases" ], "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 short data report on the molecular identification of Necator americanus in food handlers in Sudan which also serves as a 'proof of concept' for the molecular identification of hookworm species in feces. Although the report is not ‘novel’, it is new information for this region, and important information for public health officials and diagnostic laboratory scientists overall, and the data may be useful for those who study enteric parasites in humans in other regions.\n\nI recommend someone that is an expert in the molecular diagnostics of hookworm infections to carefully evaluate the methods- to the best of my knowledge the methods are sound.\n\nMajor comments:\n\nPlease include values for the proportion infected and the 95% confidence intervals for all proportions infected/detected reported in your data. I think that the discussion section could be expanded a bit more to include other studies of molecular diagnostics of hookworm and compare the prevalence and detection rates of those studies. Since in Dataset 1 you have complete positive/negative results of each technique, you may also be able to do statistical tests of agreement.\n\nIn your conclusion, you state that ‘We confirmed that the major hookworm species infecting humans in Sudan is N. americanus.’ I think that a sample size of 350 stool samples is insufficient to make that conclusion. You can say that in your sample population, you confirmed that the major hookworm species infecting humans in Sudan is N. Americans.\n\nMinor comments: First sentence Introduction- change ‘were’ to ‘are’ Change’ age of participant’ to ‘age of participants’ Fig 1- is the low molecular weight band a primer dimer?\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?\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/7-662
https://f1000research.com/articles/7-660/v1	24 May 18	{ "type": "Research Article", "title": "Influence of global and local features on parallel object identification", "authors": [ "Alvydas Šoliūnas" ], "abstract": "Background: The present study concerns parallel and serial processing of visual information, or more specifically, whether visual objects are identified successively or simultaneously in multiple object stimulus. Some findings in scene perception demonstrate the potential parallel processing of different sources of information in a stimulus; however, more extensive investigation is needed. Methods: We presented one, two or three visual objects of different categories for 100 ms and afterwards asked subjects whether a specified category was present in the stimulus. We varied the number of objects, the number of categories and the type of object shape distortion (distortion of either global or local features). Results: The response time and accuracy data corresponded to data from a previous experiment, which demonstrated that performance efficiency mostly depends on the number of categories but not on the number of objects. Two and three objects of the same category were identified with the same accuracy and the same response time, but two objects were identified faster and more accurately than three objects if they belonged to different categories. Distortion type did not affect the pattern of performance. Conclusions: The findings suggest the idea that objects of the same category can be identified simultaneously and that identification involves both local and global features.", "keywords": [ "visual perception", "object identification", "multiple objects", "global features" ], "content": "Introduction\n\nVisual information is processed in the brain in both parallel and serial. This is true not only for anatomical reason (e.g. parallel neuronal pathways from the retina; Merigan & Mounsell, 1993) but also for perceptual phenomena. An example of parallel perceptual processing is target detection in a visual search task. Features such as color, orientation, motion, spatial frequency and stereodepth are detected in parallel (Enns & Rensink, 1991; Gilchrist et al., 1997; Treisman & Gelade, 1980; Wolfe, 1994), whereas ambiguous figures and stimuli in a binocular rivalry are perceived in serial (Alais & Blake, 2004; Leopold & Logothesis, 1999). The present study concerns the identification of visual objects in multiple object stimuli and whether objects are identified in parallel or serial mode.\n\nIn scene perception, we have empirical findings that demonstrate the parallel processing of a scene category, i.e. gist perception together with perception of objects in a scene (Brandman & Peelen, 2017; Gagne & MacEvoy, 2014; Hollingworth & Henderson, 1999; Joubert et al., 2007; Joubert et al., 2008). Rousselet et al. (2002, 2004a) demonstrate parallel processing of several scenes presented simultaneously. Rousselet & colleagues (2004b) argue, based on neurophysiological findings that neurons of infero-temporal cortex with large receptive field could encode the identity of several objects in parallel and theoretically simultaneous identification of several objects is possible. On the other hand, scene identification influences identification of objects in the scene and the identification of objects influences identification of a scene (Davenport & Potter, 2004; Davenport, 2007; Joubert et al., 2007; Joubert et al., 2008; Mack & Palmeri, 2010). Such influences suppose successive perceptual processes.\n\nMuch less research has been conducted on interaction between objects presented simultaneously without scene context. Gronau et al. (2008) (see also Auckland et al., 2007; Green & Hummel, 2006) demonstrated the facilitation effect of semantically and spatially related objects on their identification in a study in which two semantically related or unrelated objects were presented in congruent or incongruent spatial relation.\n\nIf objects are identified successively, we can expect to observe a direct dependence of reaction time on the number of objects. Successive identification permits interaction between objects that could result in the nonlinear dependence of reaction time on the number of objects when reaction time depends on factors such as the similarity of objects and belongingness to the same or different categories. Our recent study demonstrated that object identification time depends much more on the number of categories than on the number of objects upon multiple object stimulus (Soliunas et al., 2018). One, two or three objects (pictures of 10 categories of man-made objects) were presented simultaneously for 100 ms and then followed by a name of a category. Subjects were asked to answer whether objects of this category were present in a stimulus. Performance accuracy and reaction time did not depend on the number of objects if the objects belonged to the same category.\n\nThe present study is further verification of the hypothesis that objects of the same category are identified simultaneously. For better control of the global features of objects, new categories were selected and new objects were produced. A shape is a possible feature of an object that could enable parallel identification. To verify this possibility, we manipulated the global and local features of objects by distorting them. We predicted that the parallel identification of objects would be observed for intact objects, but not for globally distorted objects.\n\n\nMethods\n\nAll subjects were invited to participate in the study by personal discussion. Between February and May 2015, a total of 58 volunteer students from Vilnius University agreed to took part in the experiment (44 females and 14 males; 20–22 years of age). Each subject had normal or corrected-to-normal vision and verbally confirmed that had no prior experience with psychophysical testing of a similar nature. The subjects were not informed about the specific goals of this particular experiment. All subjects signed an informed consent form approved by the Lithuanian Bioethics Committee (consent form No. ASI12, approval No. 158200-13-578-173; issued by the Vilnius Region Ethics Committee of Biomedical Research, Vilnius, Lithuania). All subjects took part in one experimental session.\n\nA total of 62 objects of 10 categories (shoe, cap, clock, ashtray, cup, table, telephone, vase, mirror, and kettle/teapot) were selected from internet search engines in such a way that the shape of an object was not the exclusive feature of particular category. When selecting objects and creating stimuli of multiple objects, seven outline shapes were taken into account: “8”-shaped, circle, ellipse, square, elongated, triangle and “L”-shaped. Objects of each category had at least three outline shapes and each outline shape had at least three categories of objects.\n\nAll objects were transformed into grayscale pictures and resized in such a way that fitted into a 100×100-pixel area. There were eight types of stimuli that varied in the number of objects (one, two or three), number of categories (one, two or three), and number of outline shapes (one or two) (Figure 1): i) “1-1” stimuli (one object); ii) “1-2” stimuli (one category, two objects of different shape); iii) “1-3” stimuli (one category, three objects of two shapes); iv) “2-2s” stimuli (two categories, two objects of the same shape); v) “2-2d” stimuli (two categories, two objects of different shapes); vi) “2-3s” stimuli (two categories, two objects of the same category and the same shape and third object of different category and different shape); vii) “2-3d” stimuli (two categories, two objects of the same category but different shapes and third object of different category but the same shape as one of the two objects of the first category); and viii) “3-3” stimuli (three categories, three objects of two shapes). In total, 10 stimuli of each type were created, giving 80 stimuli altogether (Supplementary File 1).\n\nColumns represent different types of stimulus; rows represent experimental conditions. Each individual column represents the same objects under three experimental conditions.\n\nThe objects were placed into a 200×200-pixel area around a fixation point that was located at the center of this area. Stimuli were presented at the center of screen on the white background and subjects did not see the limits of the 200×200-pixel stimulus area. Distance between the subject’s eyes and the screen was 60 cm, and consequently the angular size of the 200×200-pixel stimulus was 8°×8°. The orientation of a particular object was not constant across stimuli and could rage between −45° and +45° with respect to natural (vertical or horizontal) orientation.\n\nStimuli were presented under three experimental conditions: i) original; ii) locally distorted; and iii) globally distorted. The “original” condition corresponds to the presentation of stimuli described above. Locally distorted stimuli were created by partially masking the original stimuli with white stripes: 9-pixel-wide stripes with 9-pixel gaps (Figure 1). This procedure partially or completely eliminates some local features of objects but basically preserve the outline shape. The smaller the features, the higher the probability of elimination. Globally distorted objects were created by applying Whirl and pinch and Ripple functions in the image editor GIMP 2.8.10 (Kimball et al., 2013). The same values of these functions distort objects of different shapes to different degrees; we therefore had to apply different values of these functions to more-or-less subjectively equalize the assessed degree of distortion in different objects. Whirl and pinch values ranged from −80 to +80 for elongated and rounded shapes and from −200 to +200 for more angular shapes, and pinch amount ranged from −1 to +1. Ripple values ranged from 40 to 70. The applied global distortion procedure affects outline shape and to a lesser degree the local elements of object. The smaller the elements, the less distortion there is.\n\nTo reduce memorization of stimuli during experiment, the orientation and location of particular object in particular stimulus varied across conditions.\n\nThe experiment was performed at the Department of Neurobiology and Biophysics, Vilnius University. Experimental sessions were conducted during daytime (the precise time of the day was not controlled) in a room with natural daylight illumination.\n\nStimulus presentation and data registration were controlled by E-Prime v.2.0 (Psychology Software Tools, Inc., 2012) experiment generator running on Windows OS. Stimuli were presented on the screen of 19-inch CRT monitor running at 85 Hz frame-rate and 1024×768 resolution. The subject’s head was not fixed but they were instructed to hold the same distance (about 60 cm) from the display during experiment.\n\nBefore the experimental session, subjects performed practice session that consisted of 16 trials (two trials of each stimulus type). Only original stimuli were presented during practice.\n\nThe trial procedure of experimental session is shown in Figure 2. A fixation point was presented at the center of screen for 306 ms and the subjects were asked to keep their eyes focused on the fixation point during the test stimulus presentation. Appearance of the fixation point was followed by a 106-ms blank interval and then a test stimulus was displayed for 106 ms (i.e. for 9 frames of the CRT monitor) under “original” conditions and for 200 ms (i.e. 17 frames) under “locally distorted” and “globally distorted” conditions. The longer stimulus exposition duration under the two conditions with distorted stimuli were chosen to equalize response accuracy under all conditions. The test stimulus was followed by a 35-ms blank interval and then a masking pattern was displayed for 306 ms (we used backward masking procedure to control the time available for object identification). The masking pattern was an 8°×8° square of chaotic pattern. After 35 ms blank interval, a probe-word was presented. The probe-word was a name of a category written in lowercase Arial font, 2° height. Subjects had to decide whether an object defined by a probe-word was present or not on a given trial by pressing the “1” or “2” key on the right side of a keyboard. One half of subjects received the instruction to press the “1” for Yes and “2” for No, whereas the other half received inverse instruction. Subjects had four seconds to make their decision. The response time (the duration from onset of probe-word to the keypress event) and accuracy were recorded for each trial. The response initialized the next trial with a 106-ms delay.\n\nThe order of experimental conditions was randomized across participants. There were 60-s rest intervals between conditions. Each condition consisted of 160 trials, i.e. 80 stimuli were presented twice in random order. Altogether, 480 stimuli were presented in the experimental session, with eight types of stimuli presented randomly under each condition. The whole experimental session lasted about 30 min.\n\nThe chi-square goodness of fit test confirmed normal distribution of experimental data. The reaction time and response accuracy data were analyzed in two-way ANOVA for stimulus type (“1-1”, “1-2”, “1-3”, “2-2”, “2-3”, and “3-3”) and experimental conditions (original, locally distorted, and globally distorted). Initially, there were eight stimulus types, but as there were no statistical differences between performance for the “2-2s” and “2-2d” stimuli, we merged these results into one group “2-2”. For the same reason, we merged results of “2-3s” and “2-3d” into one group “2-3”. Newman–Keuls post hoc test was applied to assess the significance of differences between means. All statistical analysis was performed using Statistica v.7 software (StatSoft Inc., 2004).\n\n\nResults\n\nThe results of the experiment are presented in Figure 3 and Dataset 1. Two-way (stimulus type and experimental conditions) ANOVA indicated significant main effects of: stimulus type (F(5,27822) = 281.7, P < 0.0001 for reaction time (RT) data and F(5,27822) = 203.4, P < 0.0001 for response accuracy); experimental conditions (F(2,27822) = 27.7, P < 0.0001 for RT data and F(2,27822) = 49.5, P < 0.0001 for response accuracy). The interaction of the two factors was not significant (F(10,27822) = 1.7, P = 0.079 for RT and F(10,27822) = 1.2, P = 0.272 for accuracy data), which means that stimulus distortion did not change the pattern of performance that was observed for original stimuli. The significant main effect of the experimental conditions indicates that the RT was shorter (730 ms) and the accuracy was higher (86,4%) for original stimuli than for locally or globally distorted stimuli (760 ms and 80.9% for locally distorted stimuli and 762 ms and 82.1% for globally distorted stimuli), but this finding is not notable because the duration of stimulus exposition was different under different conditions and the absolute values of performance under different conditions are irrelevant. What we are interested in is the dependency of the identification of objects on the number of objects and on the number of categories.\n\nMean values are presented with 95% confidence intervals. Stimulus type: the first digit represents the number of categories in the stimulus, the second digit represents the number of objects (e.g. “1-3” represents three objects of one category).\n\nFigure 3 reveals the influence of the number of objects and the influence of the number of categories on object identification. For RT data, we can see four statistically different levels of performance (we should stress again that we compare values between “stimulus types” but not between conditions): the shortest RT is for “1-1” stimuli, with longer RTs for “1-2” and “1-3” stimuli, even longer RTs for “2-2” and “2-3” stimuli, and the longest RT for “3-3” stimuli. There is no significant difference between “1-2” and “1-3” cases (P = 0.391, 0.876, and 0.329 for “Original”, “Locally distorted”, and “Globally distorted” conditions, respectively, according to analysis using the Newman–Keuls post hoc test) and between “2-2” and “2-3” cases (P = 0.363, 0.442 and 0.472 for “Original”, “Locally distorted”, and “Globally distorted” conditions, respectively). The same four levels of performance were found under all conditions.\n\nFor accuracy data, we can see a similar pattern of performance: the highest accuracy is in “1-1” case, with lower accuracy in “1-2” and “1-3” stimulus types, even lower accuracy in the “2-2” and “2-3” stimulus type, and the lowest accuracy in the “3-3” stimulus type. Here we can see two deviations from this rule: accuracy was higher in “2-2” case than in “2-3” case under “Original” (P < 0.01) and under “Globally distorted” (P < 0.01) conditions.\n\nSummarization of the performance in relation to the number of objects and on the number of categories without differentiating experimental conditions is presented in Figure 4.\n\nMean values are presented with 95% confidence intervals.\n\nThe dependence of performance effectiveness on the number of categories is more clearly expressed than the dependence of performance effectiveness on the number of objects. For one-category stimuli, there was no difference in RT and accuracy whether two or three objects were presented. For two-category stimuli, there was no difference in RT whether two or three objects were presented, but accuracy was higher in the case of two objects. We can state that “the more categories, the poorer the performance”, but not that “the more objects, the poorer the performance”, because it depends on whether objects belong to the same or to different categories.\n\n\nDiscussion\n\nThe present study is continuation of our previous investigation (Soliunas et al., 2018) described in the Introduction, which findings suggested that the objects of the same category could be identified in parallel mode. The experiment described here further tested this hypothesis.\n\nThe first important result is the replication of the principal findings of the previous experiment, despite the fact that a different set of stimuli were used (all stimuli were newly created) and a different group of subjects took part in the experiment. These findings indicate that the identification of objects in multiple object stimuli basically depends on the number of categories present, but not on the number of objects. It further supports the suggestion that objects of the same category are identified simultaneously.\n\nThe second aim of the study was to search for the features of stimuli that could enable the parallel identification of objects of the same category, i.e. searching the “category” features that are identified in parallel. One set of stimuli had more distorted local features and the other set of stimuli had a more distorted global features. We predicted that the parallel identification of objects could be based on global features, therefore the distortion of outline shape should result in a dependency of response time on the number of objects independent of whether objects belong to the same or to different categories. The results of the experiment did not support our hypothesis. Both types of distortion had an effect only on the absolute level of performance accuracy, and to reach the same accuracy level as with intact stimuli, the exposition time for distorted stimuli was doubled. Distortion of global or local features did not change the pattern of task performance (i.e. the dependency of performance effectiveness on the number of objects and on the number of categories). At this point we can only suggest that both local and global features are used to identify the category of objects in a multiple-object environment.\n\nIt is too early to conclude that objects of the same category are identified in parallel in natural settings based on the findings of this study. Further investigations are required to test this suggestion. Here we can only speculate about the possible processes of identification of multiple objects. Our findings suggest the following scenario. As the identification of one object was faster and more accurate than identification of two objects of the same category, it is possible that the visual system first identifies one category. Additional time is required to identify other objects of this category but this could be done in parallel mode because this second stage could be regarded as “detection stage” instead of the first “identification stage”. Many studies and theories state that the detection of an object’s presence is a faster process than identification of the object’s category (Biederman, 1987; de la Rosa et al., 2011; Kobylka et al., 2017; Marr, 1982; Nakayama et al., 1995; but see Green, 1992; Grill-Spector & Kanwisher, 2005, which found no difference in the performance time between identification and detection tasks). In our case, the visual system should detect whether an object such as “shoe” (object of the first identified category) is present or not, and all shoes are detected simultaneously if they are present. Later follows the next “identification stage” when the next category is identified. It remains unclear what kind of object features are processed during the detection and identification of objects. A somewhat similar two-stage processing model of several simultaneously presented pictures was suggested by Potter & Fox (2009). They presented up to four photographs simultaneously in a rapid serial visual presentation procedure and subjects had to either detect a verbally denoted target or memorize pictures and perform a recognition test after each sequence of pictures. The two stages for visual processing suggested by the authors were: fast global processing of all pictures in a stimulus that is sufficient for target detection, and slower serial processing that is required for object recognition.\n\nIn summary, the presented experimental data support the hypothesis that visual objects of the same category are identified in parallel in multiple object stimuli. As the distortion of global or local features do not influence the performance pattern, we can suggest that both global and local features are processed during identification of object category. The number of simultaneously presented objects was restricted to three items in our experiment, therefore the further research is needed with higher number of objects.\n\n\nData and software availability\n\nDataset 1. Response time and accuracy data. \"1-1\", \"1-2\", \"2-2s\", \"2-2d\", \"1-3\", \"2-3s\", \"2-3d\", \"3-3\" are different types of stimuli where the first digit between quotation marks indicates the number of categories and the second digit indicates the number of objects. Original, original conditions; locally d., locally distorted conditions; globally d., globally distorted conditions; RT, reaction time; %, percentage accuracy. DOI: 10.5256/f1000research.14468.d204177 (Šoliūnas, 2018).\n\nFree alternatives for E-prime software: PsychoPy (Peirce, 2007); DXMD (Forster & Forster, 2003).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nSupplementary File 1. Stimuli used in the experiment.\n\nClick here to access the data.\n\n\nReferences\n\nAlais D, Blake R, eds.: Binocular Rivalry. MIT Press: Cambridge. 2004. Reference Source\n\nAuckland ME, Cave KR, Donnelly N: Nontarget objects can influence perceptual processes during object recognition. Psychon Bull Rev. 2007; 14(2): 332–337. PubMed Abstract \| Publisher Full Text\n\nBiederman I: Recognition-by-components: A theory of human image understanding. Psychol Rev. 1987; 94(2): 115–147. 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PubMed Abstract \| Publisher Full Text\n\nRousselet GA, Thorpe SJ, Fabre-Thorpe M: Processing of one, two or four natural scenes in humans: the limits of parallelism. Vision Res. 2004a; 44(9): 877–94. PubMed Abstract \| Publisher Full Text\n\nRousselet GA, Thorpe SJ, Fabre-Thorpe M: How parallel is visual processing in the ventral pathway? Trends Cogn Sci. 2004b; 8(8): 363–70. PubMed Abstract \| Publisher Full Text\n\nŠoliūnas A: Dataset 1 in: Influence of global and local features on parallel object identification. F1000Research. 2018. Data Source\n\nSoliunas A, Pleskaciauskas A, Daktariunas A: Multiple object categorization and effect of spatial frequencies. PeerJ Preprints. 2018; 6: e26666v1. Publisher Full Text\n\nStatSoft, Inc., Tulsa, USA: [Statistica 7.0]. 2004. Reference Source\n\nTreisman AM, Gelade G: A feature-integration theory of attention. Cogn Psychol. 1980; 12(1): 97–136. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "34363", "date": "05 Jun 2018", "name": "Dan McCarthy", "expertise": [ "Reviewer Expertise Form perception", "motion perception", "attention", "visually-guided action", "synesthesia" ], "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 investigated whether multiple objects are processed serially or in parallel by testing the impact of global and local feature distortions on identification performance. Specifically, participants identified whether an object belonging to a particular category was present in a display consisting of 1-3 objects. Local distortion was applied by masking images with stripes masking the background color of the display, thereby occluding local object features while preserving the global shape outline. Global distortion was accomplished by warping objects in an image editor, allowing full view of the object despite deformations in individual features and the global object outline.\nResults indicate that such distortion led to slowed responses and decreased accuracy in identifying an object category, but this is attributed on differences in the exposure duration (~2x longer viewing time for distorted stimuli to achieve similar performance to originals). Increasing the number of categories present in a display was associated with lower accuracy and slower response times when 2 or more objects were shown. No reaction time differences were observed in the key contrast between the 2 or 3 object displays, yet accuracy was higher with 2 vs. 3 objects in the 2 category case.\nBased on these findings, the author argues that multiple objects can be identified simultaneously, but only when they belong to the same category. The lack of performance differences between locally and globally distorted stimuli was interpreted as evidence that object identification relies on both local and global features.\nWhile it is written clearly and the question is interesting as other work indeed suggests the plausibility of multiple object identification in parallel, I have statistical and methodological concerns related to the interpretation of these results that I list below:\n1. A two-way ANOVA is not the appropriate statistical test given my understanding that all participants completed the same task. A 2-way repeated measures ANOVA should be used instead. This may explain the extremely large F-values and within-groups degrees of freedom (i.e., DFwithin = 27822 for all main effects and the interaction). I am unsure how this value was obtained. The correct DFwithin should be 285 for stimulus type ((6 groups - 1)(58 participants - 1) = 557 = 285), 114 for distortion ((3 groups - 1)(58 participants - 1) = 257 = 114), and 570 for the interaction (5257 = 570). This test should be redone correctly and accurately reported to confirm the reported results hold.\n2. Another concern is that the primary conclusion for parallel object identification within a single category is based on a failure to reject the null. No power analysis is reported to indicate the chance of a Type II error. A powerful approach to address evidence in favor of the null would be to use Bayes Factors (for examples, see Wagenmakers, 20071; Jarosz & Wiley, 20142). This can be easily implemented in JASP (Love et al., 20153) - freely available, user friendly software that the author could use to conduct a Bayesian version of the two-way repeated measures test discussed above that would allow a direct test of the likelihood that the null hypothesis (no RT difference between identifying 2 or 3 objects of the same category) is a better model of the data than the alternative (a difference). Jarosz & Wiley (2014) is an excellent resource for how to interpret the results of such a test and what strength of evidence favoring either hypothesis may be.\n3. The use of occluding bars for local distortion is somewhat odd given that the same 'whirl and pinch' effects could be applied to the interior of objects without distorting the global boundary. A brief description of why these disparate methods were chosen or examples in previous literature would be helpful for the reader.\n\nI look forward to seeing a revised version of this work once these concerns have been properly addressed.\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": "35054", "date": "15 Jun 2018", "name": "Thiago Monteiro de Paiva Fernandes", "expertise": [ "Reviewer Expertise Visual perception", "Cognition", "Psychophysics" ], "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 reports the influence of global and local features on parallel object identification using one, two or three visual objects. Author's efforts to conduct a proper study needs to be mentioned/recognised. The author provided a clear and testable hypothesis and the article is straightforward.\n\n1) The introduction is somewhat unfocused. The author rapidly switch from discussing theoretical and physiological aspects of visual perception, but do not strongly compare their assumptions with alternative theories. Although it is already brief, I reckon the Introduction could be made more concise and should be edited to improve flow.\n\n- There are a considerable number of obvious and/or dubious statements (e.g., Visual information is processed in the brain in both parallel and serial what?; Features such as color, orientation, motion, spatial frequency and stereodepth are detected in parallel). Although it is true that we can not ignore the complexity of retinal information, seems like the author ignored the boundary completion. When one take part in some assumption (e.g., parallel; apologise if I got it wrong), it is fundamental that both sides (parallel vs serial) could be briefly discussed.\n\n- The author reported some old references to support assumptions. Some of them appear to have been poorly chosen/incorrect. Maybe the author could update them. I reckon this will strengthen the arguments.\n\n- Scene context discussion (one or two sentences) could be interesting to improve Introduction.\n\n2) Overall, some parts of the article is difficult to understand due to the insufficient English language quality (examples are listed below) and many typographical errors throughout the text. I suggest that the authors enlist an editor/colleague to improve the English.\n\nEnglish quality examples:\n\n\"Visual information is processed in the brain in both parallel and serial.\" - What? Processing? Yes, I understood, but seems like we need a complement.\n\n\"Much less research\" - What about \"Research on interaction between objects... are underreported...\" ?\n\n\"For better control of the global features of objects, new categories were selected and new objects were produced\"\n\n- It is not clear what the authors' intent is in this paragraph. Further, it is not clear if the authors are making a prediction if producing new objects would affect parallel identification.\n\n3) The absence of relevant information in the Methods section is worrisome. There is a lack of detail about study procedures.\n\n- The subjects were assessed for neuropsychiatric disorders?\n\n- The subjects were assessed for substance use?\n\n- \"normal or corrected-to-normal\"; Do you mean 20/20? Using eye chart? Freiburg visual acuity?\n\n- Don't you think that the sample characteristics biased the results? (e.g., only young subjects; significant gender differences; menstrual cycle influence; cognitive performance). If you disagree, please update Subjects subsection.\n\n- \"All of the objects were transformed into grayscale pictures and resized in such a way ... 100x100-pixel\" I see your point. However, the readers also need to understand your point. Why grayscale? Why 100 x 100?\n\n- Luminance of the monitor screen?\n\n- Why 60 cm? This was based on previous studies?\n\n- Why you chose 106-ms blank interval? Seems like an odd number if you do not explain.\n\nFurther, in my opinion some of methods could be reorganised into more relevant sub-sections.\n\n4) Statistical analysis needs to be reformulated. I reckon the Bayes Factor would be an interesting approach. Nevertheless, I recognise this may take a long time to update. My main concern is about the ANOVA. I see your point for merging results (2-2s and 2-2). However, it seems worrisome. I suggest to you performing the analysis without merge the results (multivariate analysis).\n\n- Why the use of chi-square (fit test) for normal distribution?\n\n- Why the Newman-Keuls post-hoc (and not the REGWQ)?\n\n- Effect size? Confidence Interval? Please provide such additional information.\n\nI agree with the other reviewer about the possibility of Type I and/or Type II error. Thus, you will need to reorganise this section.\n\n5) I am unsure what implications are to be drawn from the study at present. I mean, you need to better address this on Discussion.\n\n- Although the author can not drawn physiological conclusions, it should be - at least - mentioned.\n\n- You supported the hypothesis \"that visual objects of the same category are identified in parallel...\" I see your point. However, this needs to be better addressed. The Discussion should have more references.\n\nI am providing some references to help in the organisation.\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?\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? Yes", "responses": [] }, { "id": "35055", "date": "25 Jun 2018", "name": "Denis V. Yavna", "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 is aimed to answer whether the identification of objects and its category in an multi-object stimuli are performed serially or in parallel. It is shown that the number of categories of simultaneously presented objects, but not the number of objects per se, affects the time and accuracy of the identification of a category. The author demonstrates that if several objects belong to a smaller number of categories, then the categories are easier to identify than when several objects belong to a larger number of categories. It is assumed that objects of one category are identified in parallel mode.\nThe main problem of this study is that it is much more possible to guess the right answer when there are more than one image of one category that are presented simultaneously comparing to stimuli that consist of images of different categories. Suppose there are 3 objects belonging to the same category. If an observer identifies at least one object, they are more likely to give the correct answer than in a situation where there are two categories: a priori, they have less chance to detect an object of one of these categories. It seems that accounting for a priori probability is necessary. The simplest thing that comes to mind is subtraction of the total probability of random guessing from an empirical estimate of the probability for each type of stimulus (1-1, 1-2, etc).\nI agree with Dan McCarthy concerning the issue of the statistical analysis used by the author. Probably, for a more correct (and powerful in a statistical sense) comparison of test results performed through repeated testing of observers in similar tasks, it is necessary to apply a method that gives special attention to differences in performance in different tasks and not to the individual differences in speed and accuracy of identification; e. g. repeated measures ANOVA is more preferable. And of course, in the presence of conclusions based on the adoption of the null hypothesis, power analysis is desired. Probably this1 work containing tables with power estimations for the 3x6 design, will be useful for author.\nI also would like to note that the use of the chi-square test to check the normality of distribution is not recommended by many authors (e. g. 2 p. 94]); but this note is not fundamental.\nUndoubtedly, the work is of great interest and should be indexed, but a significant revision is required.\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": "35056", "date": "28 Jun 2018", "name": "Mohd Izzuddin Hairol", "expertise": [ "Reviewer Expertise visual perception", "visual psychophysics" ], "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 investigated whether identification performance of objects are performed serially or in parallel. The author shows that reaction time and accuracy of object identification are affected by the number of categories of objects, but not the number of objects. There are a few issues that the author need to address.\n\nParts of the Introduction seem incompletely discussed/elaborated. For example, in para 3, the elaboration of the previous research would be better if they were related back clearly to the purpose of the current study.\n\nInsufficient details in Methods\nWhat do the authors mean by corrected-to-normal vision? How was it assessed?\n\nWhat was the size of each pixel? Since the stimulus size was restricted to 100x100 pixels, the angular size of the objects, particularly if they had details that might be important for recognition, might have influence response accuracy.\n\nAmong the 10 categories of objects that were chosen, were they tested to be equally identifiable? Some objects might be harder to identify than others, therefore could affect reaction time and accuracy.\n\nThe author mentioned that “the longer stimulus exposition duration under the two conditions with distorted stimuli were chosen to equalize response accuracy under all conditions” (page 3). Well if response accuracy had been equalize then of course one would not find any difference in duration time between original, locally-distorted and globally distorted objects?\n\nA repeated-measures ANOVA would be more suitable to analyse the data.\n\nThere are several English language issues that the author need to address, as have been pointed out by other reviewers. A glaring example is the first sentence in the article: “Visual information is processed in the brain in both parallel and serial.” Parallel and serial WHAT?\nThe implications of the study need to be better discussed.\n\nThe current study is undoubtly of interest but all of these concerns need to be addressed.\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": [] } ]	1	https://f1000research.com/articles/7-660
https://f1000research.com/articles/7-658/v1	24 May 18	{ "type": "Software Tool Article", "title": "autoHGPEC: Automated prediction of novel disease-gene and disease-disease associations and evidence collection based on a random walk on heterogeneous network", "authors": [ "Duc-Hau Le", "Trang T.H. Tran", "Trang T.H. Tran" ], "abstract": "Identification of novel disease-gene and disease-disease associations is an important task in biomedical research. Recently, we have developed a Cytoscape app, namely HGPEC, using a state-of-the-art network-based method for such task. This paper describes an upgrading version of HGPEC, namely autoHGPEC, with added automation features. By adding these functions, autoHGPEC can be used as a component of other complex analysis pipelines as well as make use of other data resources. We demonstrated the use of autoHGPEC by predicting novel breast cancer-associated genes and diseases. Further investigation by visualizing and collecting evidences for associations between top 20 ranked genes/diseases and breast cancer has shown the ability of autoHGPEC.", "keywords": [ "Cytoscape app", "Automation features", "CyREST", "R", "Disease-gene association", "Disease-disease association", "Random walk with restart algorithm", "Heterogeneous network", "Gene prioritization", "Disease prioritization" ], "content": "Introduction\n\nOne of the challenging tasks in biomedicine is to prioritize candidate genes and diseases by the degree of their relevance to a disease of interest. This is the starting point to identify novel disease-gene and disease-disease associations. A large number of computational methods including network- and machine learning-based ones have been proposed for such a task1,2. State-of-the-art network-based methods often integrate diseases and genes together to form a heterogeneous network, then a propagation algorithm is applied to exploit the similarity between diseases/genes and known disease-gene associations to predict novel associations3–7. Some tools have been also developed to facilitate the use of the state-of-the-art methods. However, most of them only focus on predicting novel disease-gene associations8–10, including some tools which were developed as apps of Cytoscape11. Recently, we have developed a Cytoscape app, HGPEC12, to predict both disease-gene and disease-disease associations based on a state-of-the-art method on a heterogeneous network of diseases and genes3. HGPEC was shown to be better than two other network-based Cytoscape apps for prediction of novel disease-gene associations, GPEC13 and PRINCIPLE14 in terms of prediction performance12. In addition, HGPEC can prioritize candidate genes of diseases without known molecular basis and collect evidence to support novel predictions from various data resources such as Gene Ontology15, Disease Ontology16, KEGG pathway17, GeneRIF18, PubMed19, protein complexes20 and OMIM21. Being developed as an app of Cytoscape, HGPEC can exploit advanced features of Cytoscape such as data visualization and integration. However, Cytoscape is a desktop-based tool, thus HGPEC cannot link to other analysis tools such as R and Python flexibly. Therefore, this also limits the use of HGPEC because it cannot be used automatically as a component of a complex analysis pipeline in these tools. In addition, this prevents Cytoscape from integrating data from other data resources. Recently, automation features have been added to Cytoscape to facilitate those tasks.\n\nIn this study, we upgrade HGPEC by adding automation features into it and name the new app as autoHGPEC. Basically, autoHGPEC has the same functions as HGPEC. However, these functions can be called by both CyREST functions and commands, thus can be called from external environments. To use autoHGPEC, a heterogeneous network of diseases and genes composing of a disease similarity network, a gene/protein network and known disease-gene associations has to be given. Then, a disease of interest must be selected from the disease similarity network. After that, the disease and its known associated genes (if any) are used as training/seed data. A set of candidate genes then has to be defined by selecting from the gene network or chromosome. These candidate genes and all remaining diseases are then ranked by a RWRH-based method (see the Methods section). Finally, users can select top ranked genes/diseases for further analyses such as visualization and evidence collection. We show the ability of autoHGPEC in predicting novel genes and diseases associated with breast cancer.\n\n\nMethods\n\nautoHGPEC was implemented using a ranking algorithm, random walk with restart on a heterogeneous network (RWRH)12. Briefly, this network-based algorithm propagates the disease information embedded in a disease of interest and its known associated genes (also known as seed/training nodes) to other diseases and genes in the heterogeneous network. This propagation is performed by random walking from the seed nodes. At each node, the random walker goes to adjacency nodes or goes back to the seed nodes with a prior probability. This process is repeated iteratively until a steady-state is reached. A score assigned to each node at this state represents the degree of relevance to the seed nodes, thus relevance to the disease of interest. Finally, candidate genes and diseases are ranked by the scores and top ranked candidates can be selected as promising genes and diseases for further investigation.\n\nautoHGPEC is an upgrading version of HGPEC12 with added automation features. Therefore, main functions such as prioritization, visualization and evidence collection of HGPEC were kept. In addition, as in HGPEC, a number of databases were preinstalled in autoHGPEC to facilitate the use of this app. These include disease similarity networks, gene/protein networks and known disease-gene associations as well as annotation data such as Gene Ontology15, Disease Ontology16, KEGG pathways17, GeneRIF18, and protein complexes20. However, users can also select other networks by themselves. In order to provide automation features for HGPEC, we first refactor source code of HGPEC to implement Cytoscape Tunable annotations to replace control panels of HGPEC in the west by a menu system. Therefore, all the functions of HGPEC are accessed through the menu system. In addition, the workflow of HGPEC is exposed to the users by using CyREST Command API (which can be followed in Swagger UI under the menu autoHGPEC). The CyREST API is developed with appropriated functions as well. Thus, the result of each step in the workflow can be passed on to the caller for further analysis in R or Python in JSON format.\n\nautoHGPEC is designed to predict novel disease-gene and disease-disease associations and evidence collection based on a random walk on heterogeneous network with added automation features. Therefore, it operates in the same workflow as in HGPEC12. However, in addition to desktop-based Cytoscape though the menu system, its functions can be called using CyREST Command API and from other analysis tools such as R. Figure 1 show the workflow of autoHGPEC in three running environments (see user manual in Supplementary File 1). As an app of Cytoscape with automation features, autoHGPEC can be run on any computer which satisfies the minimal requirements to run Cytoscape.\n\nTo demonstrate functions of autoHGPEC with automation features, we showed its ability in predicting novel genes and diseases associated with breast cancer (OMIM ID: 114480). Here, we briefly describe this case study by following the 5-step workflow in Figure 1 (see user manual in Supplementary File 1 for more detail):\n\n- First, a heterogeneous network of genes and diseases was constructed by connecting a preinstalled disease similarity network (i.e., Disease_Similarity_Network_5) including 5,080 diseases and 19,729 interactions, a preinstalled human protein interaction network (i.e., Default_Human_PPI_Network) including 10,486 genes and 50,791 interactions, and known disease-gene associations collected from OMIM21. This step can be accomplished by following commands from within R:\n\n> commandRun('autoHGPEC step1_construct_network DiseaseGene=\"Disease-gene from OMIM\" diseaseNetwork=\"Disease_Similarity_Network_5\" geneNetwork=\"Default_Human_PPI_Network\"')\n\n- Second, breast cancer (OMIM ID: 114480) was selected for investigation. This disease is known to be associated with 21 genes, which are also available in the human protein interaction network. Then, the training set was built with these genes and the disease of interest. We can run two following commands within R for this task:\n\n> commandRun('autoHGPEC step2_1_select_disease diseaseName=\"breast cancer\"')\n\n> commandRun('autoHGPEC step2_2_create_training_list diseaseTraining=\"MIM114480\"')\n\n- Third, we selected all of 10,465 remaining genes in the protein interaction network as candidate genes. This option can be done by following command:\n\n> commandRun('autoHGPEC step3_PCG_allRemaining')\n\n- Fourth, all genes and diseases in the heterogeneous network are ranked by applying the RWRH-based method with back-probability, jumping probability and subnetwork importance weight were set to 0.5, 0.6 and 0.7, respectively. The following command can be used to accomplish this task:\n\n> commandRun('autoHGPEC step4_prioritize backProb=0.5 jumpProb=0.6 subnetWeight=0.7')\n\n- Finally, we visualized and collected evidence for the associations between 20 highly ranked candidate genes/diseases and breast cancer. The users must highlight the diseases and genes of their interest in the corresponding network. These tasks can be performed using two following commands, respectively:\n\n> commandRun('autoHGPEC step5_2_visualize')\n\n> commandRun('autoHGPEC step5_1_search_evidences')\n\nVisualization results (Figure 2a and b) show that most of the top ranked candidate genes are directly connected to known breast cancer-associated genes. In addition, highly ranked candidate diseases are directly connected to either known/training genes or the disease of interest. For evidence collection, we annotated and searched evidence for promising associations between the top ranked candidate genes/disease and breast cancer. Evidence collection results showed that each of the promising associations is supported by at least two data sources. More detail about interpretation on the results of visualization and evidence collection for these associations can be found in the HGPEC study12. Beside the fact is that almost commands of autoHGPEC return results in JSON format, the results of autoHGPEC is revealed via CyREST API as well (menu Help/Automation/CyREST Api). For example, the command in R, commandRun('autoHGPEC step2_1_select_disease diseaseName=\"breast cancer\"'), in Step 2 can be performed directly by CyREST API with the request URL http://localhost:1234/autohgpec/v1/selectDisease/breast%20cancer (this URL is available after successfully constructing the heterogeneous network in Step 1). Then, it returns a list of OMIM IDs associated with “breast cancer” in JSON format as follows:\n\n[\n\n{\n\n\"name\": \"BREAST CANCER 1 GENE; BRCA1\",\n\n\"DiseaseID\": \"MIM113705\",\n\n\"MedGenCUI\": \"\"\n\n},\n\n{\n\n\"name\": \"BREAST CANCER\",\n\n\"DiseaseID\": \"MIM114480\",\n\n\"MedGenCUI\": \"C0346153\",\n\n\"AssociatedGenes\": \"5888, 3845, 83990, 8493, 580, 841, 3161, 7517, 9821, 79728, 5245, 5002, 672, 675, 5290, 11200, 207, 472, 4835, 999, 7157, 8438\"\n\n},\n\n{\n\n\"name\": \"BREAST-OVARIAN CANCER, FAMILIAL, SUSCEPTIBILITY TO, 1; BROVCA1\",\n\n\"DiseaseID\": \"MIM604370\",\n\n\"MedGenCUI\": \"C2676676\",\n\n\"AssociatedGenes\": \"4978, 2956, 672, 5290, 207, 5071\"\n\n},\n\n{\n\n\"name\": \"BREAST CANCER ANTIESTROGEN RESISTANCE 3; BCAR3\",\n\n\"DiseaseID\": \"MIM604704\",\n\n\"MedGenCUI\": \"\"\n\n}\n\n]\n\nTherefore, users can easily call this CyREST API and use this result in their workflow as they need.\n\n(a) Topological relationships between highly ranked candidate genes and known breast cancer-associated genes. (b) Topological relationships between highly ranked candidate diseases and breast cancer and its known associated genes. Note that: For diseases, nodes in rhombus and rectangle shapes are breast cancer and candidate diseases, respectively. For genes, nodes in triangle and octagon shapes are known breast cancer-associated genes and candiate genes, respectively. Nodes with high rankings are in red, relative high are in pink, medium are in white and light green, low are in green.\n\n\nDiscussion and conclusions\n\nRandom walk with restart algorithm on heterogeneous network of diseases and genes was shown as a state-of-the-art method for predicting novel disease-gene and disease-disease associations compared to other network-based algorithms3,12. However, its prediction performance highly depends on the used heterogeneous network, which is a combination of a disease similarity network and a gene/protein interaction network and known disease-gene associations. Indeed, a study showed that the prediction performance can be improved by using a gene ontology-based gene similarity network instead of using the human protein interaction network22. In addition, we have recently shown that using the disease similarity network constructed by Human Phenotype Ontology23 improved the prediction performance of disease-associated genes24 as well as disease-associated non-coding RNAs25,26. Therefore, to facilitate the use of the similarity networks of diseases/genes, we enable user to provide these networks by themselves. For gene/protein network, user can import the network from various molecular interaction data sources or from other analysis pipelines. Similarly, disease similarity networks can be inputted from other analysis tools such as DOSim27 and HPOSim28. Moreover, the ranked candidate genes can be used as inputs of other annotation and enrichment toolkits to support more about their associations with the disease of interest such as DAVID29 and GSEA30. Taken together, with added automation features, autoHGPEC can be more useful and reached by a wider range of users.\n\nIdentification of novel disease-gene and disease-disease associations is an important task in biomedical research. Recently, we have developed a Cytoscape app, namely HGPEC, using a state-of-the-art network-based method for such task. This paper describes an upgrading version of HGPEC, namely autoHGPEC, with added automation features. By adding these functions, autoHGPEC can be used as a component of other complex analysis pipelines as well as make use of other data resources. We demonstrated the use of autoHGPEC by predicting novel breast cancer-associated genes and diseases. Further investigation by visualizing and collecting evidences for associations between top 20 ranked genes/diseases and breast cancer has shown the ability of autoHGPEC.\n\n\nSoftware and data availability\n\n1. autoHGPEC on Cytoscape Apps: http://apps.cytoscape.org/apps/autohgpec\n\n2. User manual can be downloaded at https://sites.google.com/site/duchaule2011/bioinformatics-tools/autohgpec\n\n3. autoHGPEC can be run from within R using RCy3 (https://www.bioconductor.org/packages/release/bioc/html/RCy3.html)\n\n4. Source code: https://github.com/trangtran86/autoHGPEC\n\n5. Archived source code as at time of publication: http://doi.org/10.5281/zenodo.122852131\n\n6. License: MIT\n\nAll prerequisite data are already included in the apps. Refer to the user manual (Supplementary File 1) for other additional annotation data such as Gene Ontology.", "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: autoHGPEC user manual.\n\nClick here to access the data.\n\n\nReferences\n\nBarabási AL, Gulbahce N, Loscalzo J: Network medicine: a network-based approach to human disease. Nat Rev Genet. 2011; 12(1): 56–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang X, Gulbahce N, Yu H: Network-based methods for human disease gene prediction. Brief Funct Genomics. 2011; 10(5): 280–293. PubMed Abstract \| Publisher Full Text\n\nLi Y, Patra JC: Genome-wide inferring gene-phenotype relationship by walking on the heterogeneous network. Bioinformatics. 2010; 26(9): 1219–1224. PubMed Abstract \| Publisher Full Text\n\nChen Y, Jiang T, Jiang R: Uncover disease genes by maximizing information flow in the phenome-interactome network. Bioinformatics. 2011; 27(13): i167–i176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuo X, Gao L, Wei C, et al.: A computational method based on the integration of heterogeneous networks for predicting disease-gene associations. PLoS One. 2011; 6(9): e24171. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLe DH, Nguyen MH: Towards more realistic machine learning techniques for prediction of disease-associated genes. In: Proceedings of the Sixth International Symposium on Information and Communication Technology; Hue City, Viet Nam. 2833269: ACM. 2015; 116–120. Publisher Full Text\n\nLe DH, Xuan Hoai N, Kwon YK: A Comparative Study of Classification-Based Machine Learning Methods for Novel Disease Gene Prediction. In: Knowledge and Systems Engineering. Edited by Nguyen V-H, Le A-C, Huynh V-N, Springer International Publishing; 2015; 326: 577–588. Publisher Full Text\n\nOti M, Ballouz S, Wouters MA: Web tools for the prioritization of candidate disease genes. In: In Silico Tools for Gene Discovery. Methods Mol Biol. 2011; 760: 189–206. PubMed Abstract \| Publisher Full Text\n\nTranchevent LC, Capdevila FB, Nitsch D, et al.: A guide to web tools to prioritize candidate genes. Brief Bioinform. 2011; 12(1): 22–32. PubMed Abstract \| Publisher Full Text\n\nMoreau Y, Tranchevent LC: Computational tools for prioritizing candidate genes: boosting disease gene discovery. Nat Rev Genet. 2012; 13(8): 523–536. PubMed Abstract \| 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\nLe DH, Pham VH: HGPEC: a Cytoscape app for prediction of novel disease-gene and disease-disease associations and evidence collection based on a random walk on heterogeneous network. BMC Syst Biol. 2017; 11(1): 61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLe DH, Kwon YK: GPEC: A Cytoscape plug-in for random walk-based gene prioritization and biomedical evidence collection. Comput Biol Chem. 2012; 37: 17–23. PubMed Abstract \| Publisher Full Text\n\nGottlieb A, Magger O, Berman I, et al.: PRINCIPLE: a tool for associating genes with diseases via network propagation. Bioinformatics. 2011; 27(23): 3325–3326. PubMed Abstract \| Publisher Full Text\n\nAshburner M, Ball CA, Blake JA, et al.: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25(1): 25–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchriml LM, Arze C, Nadendla S, et al.: Disease Ontology: a backbone for disease semantic integration. Nucleic Acids Res. 2012; 40(Database issue): D940–D946. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanehisa M, Goto S, Furumichi M, et al.: KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res. 2010; 38(Database issue): D355–D360. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitchell JA, Aronson AR, Mork JG, et al.: Gene indexing: characterization and analysis of NLM's GeneRIFs.AMIA Annu Symp Proc. 2003; 2003: 460–4. PubMed Abstract \| Free Full Text\n\nSayers EW, Barrett T, Benson DA, et al.: Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2011; 39(Database issue): D38–D51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuepp A, Brauner B, Dunger-Kaltenbach I, et al.: CORUM: the comprehensive resource of mammalian protein complexes. Nucleic Acids Res. 2008; 36(Database issue): D646–D650. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmberger J, Bocchini CA, Scott AF, et al.: McKusick's Online Mendelian Inheritance in Man (OMIM). Nucleic Acids Res. 2009; 37(Database issue): D793–D796. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang R, Gan M, He P: Constructing a gene semantic similarity network for the inference of disease genes. BMC Syst Biol. 2011; 5 Suppl 2: S2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKöhler S, Doelken SC, Mungall CJ, et al.: The Human Phenotype Ontology project: linking molecular biology and disease through phenotype data. Nucleic Acids Res. 2014; 42(Database issue): D966–D974. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLe DH, Dang VT: Ontology-based disease similarity network for disease gene prediction. Vietnam Journal of Computer Science. 2016; 3(3): 197–205. Publisher Full Text\n\nLe DH: Disease phenotype similarity improves the prediction of novel disease-associated microRNAs. In: Information and Computer Science (NICS), 2015 2nd National Foundation for Science and Technology Development Conference on: 16–18 Sept. 2015. 2015; 76–81. Publisher Full Text\n\nLe DH, Dao LTM: Annotating diseases using human phenotype ontology improves prediction of disease-associated long non-coding RNAs. J Mol Biol. 2018; pii: S0022-2836(18)30401-7. PubMed Abstract\n\nLi J, Gong B, Chen X, et al.: DOSim: An R package for similarity between diseases based on Disease Ontology. BMC Bioinformatics. 2011; 12(1): 266. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeng Y, Gao L, Wang B, et al.: HPOSim: an R package for phenotypic similarity measure and enrichment analysis based on the human phenotype ontology. PLoS One. 2015; 10(2): e0115692. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDennis G Jr, Sherman BT, Hosack DA, et al.: DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003; 4(9): R60. 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): 15545–15550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\ntrangtran86: trangtran86/autoHGPEC: First commit (Version 1.0). Zenodo. 2018. Data Source" }	[ { "id": "36572", "date": "07 Aug 2018", "name": "Tin Nguyen", "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 aims to address the challenging task of trying to prioritize candidate genes and diseases based on their degree of relevance within a known heterogenous network of disease-gene and disease-disease associations. A previous iteration of this Cytoscape app called HGPEC employs a network learning-based method only for disease-gene associations however now it has been upgraded to autoHGPEC with added automation features, the flexibility to be integrated in more complex analysis pipelines and the ability to take input other data resources. Another addition is that autoHGPEC predicts not only disease-gene associations but disease-disease assocations as well. This paper details the application of autoHGPEC in predicting novel breast cancer-associated genes and diseases with increased automation and flexibility.\nI commend the authors in the presentation of their work. The paper was logical, concise and easy to read. Below are my comments. I hope it will benefit them well.\n\nMajor comments:\nIt is mentioned that the app is highly dependent on a used heterogenous network, gene/protein interaction networks and known disease-gene associations. Firstly, clarify how these networks are provided and what exactly they are. Secondly, perhaps using gene ontology-based gene similarity networks could be better. This is crucial, as there could be post translational modification effects to be explored. However, with this said, the paper claims HGPC can prioritize candidate genes without knowing its molecular basis. Reconsider the reasoning behind this claim.\nHow is the input and output data reliable if we don’t know the molecular basis? Perhaps this is a dangerous statement, as there’s now lots of computational tools providing evidence to the pertinence of post translational modification effects such as methylation, acetylation, etc. Be careful how this is stated. It has current and will have future ramifications. At least provide an explanation as to why knowledge of this isn’t needed.\n\nIf autoHGPEC performs better than GPEC and PRINCIPLE, present data on it. Where’s the comparison between the three and what is the criteria for ranking them?\n\nHas a comparison of results for when a set of candidate genes are selected from a gene network or chromosome been done? This is a flexibility-based component of the app however testing these cases could give evidence for which is more reliable and in what circumstances.\n\nMinor comments\n“Recently, we have developed a Cytoscape app, HGPEC, to predict both disease-gene and disease-disease associations based on a state-of-the-art method on a heterogeneous network of diseases and genes.” Be specific. I understand what the paper is trying to say here, with “state of the art” meaning either network or machine learning based approaches however it took a few times to get a clear understanding. Mention how pertinent these approaches are but afterwards, mention which state of the art approach is being used. It will get the point across quickly without introducing any confusion.\n\nTechnical and grammatical writing errors:\n- “…we first refactor source code of HGPEC to implement Cytoscape Tunable annotations to replace control panels of HGPEC in the west” – - “Beside the fact is that almost commands of autoHGPEC return results in JSON format…” - “autoHGPEC is an upgradED version of HGPEC…” - “For gene/protein networkS, user can import the network from various molecular interaction”\nThere are more throughout the paper and especially the supplementary document. Read through and correct carefully. Pay attention to your font formatting and spacing to keep things consistent.\n\nChange the node colors on page 8 of the supplementary document. It is a little confusing to see red as top ranked, green as bottom ranked and white and light-green as middle ranked. Maybe provide a legend.\n\nBottom of page 10 – change column names for abbreviation for association. Pick something other than ass. Maybe assoc.\n\nOverall, good job. Two crucial benefits to the new app is that it can take data in from multiple sources as opposed to only one source – possibly lowering the chance of error and bias, and that it lets you integrate it with R and Python, allowing for integration as a component of more complex analysis pipelines. However, it seems this app can only be used if known disease-gene/protein associations and disease similarity networks are given. Why doesn’t the paper mention protein interactions any further? Specifically, what pertinent information is being taken away from these said protein interactions?\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": "36046", "date": "28 Aug 2018", "name": "Thanh Le Van", "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\nSummary:\nThe paper \"autoHGPEC: Automated prediction of novel disease-gene and disease-disease associations and evidence collection based on a random walk on heterogeneous network\" presents an enhanced implementation of HGPEC, the previous work of one of the co-authors of the paper. The new implementation allows users to integrate the data analysis steps in Cytoscape with other data analysis pipelines in R or CyREST API. Indeed, this new feature would be useful as users now can take the advantages of the network-based data analysis and visualization in Cytoscape as well as the power of statistical data analysis of R, for example.\nBelow are my detail comments:\nIs the rationale for developing the new software tool clearly explained?\nIn my opinion, the \"automatic features\" is not well explained in the paper. The first place where the authors introduce the concept of \"automatic features\" is the last sentence of the first paragraph in the Introduction section. However, there is no further explain of this concept. Hence, it is very easy for people in the machine learning community to be confused with the concept of automatic feature selection in the automated machine learning field.\nTo clear the possible confusion, we can do two things: 1) add a citation of the paper/website where Cytoscape orginally introduce this concept; 2) briefly explain how Cytoscape provides this type of feature and how HGPEC can leaverage the facilities provided by Cystocape.\n\nIs the description of the software tool technically sound?\nYes\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?\nThe user manual is quite detail. However, there are rooms for improvements of the presentation, for example, the space between pictures and paragraphs, and the ident of paragraph are not always consistent and pleasant to read. I highly recommend to use latex to produce the mamual.\nThere is a Vietnamese sentence on page 10 of the manual, which should be removed.\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool?\nPartly\n- Please add a citation when mentioning that breast cancer is known to be associated with 12 genes (first paragraph, page 5) - Please briefly explain why the results of the demonstration make sense\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article?\nYes\n\nOverall, the author should have further explain: what is automatic features and why it is worthy of investigation.\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": [] } ]	1	https://f1000research.com/articles/7-658
https://f1000research.com/articles/7-531/v1	02 May 18	{ "type": "Software Tool Article", "title": "Automation of ReactomeFIViz via CyREST API", "authors": [ "Fred Loney", "Guanming Wu", "Fred Loney" ], "abstract": "Pathway- and network-based approaches project seemingly unrelated genes onto the context of pathways and networks, enhancing the analysis power that cannot be achieved via gene-based approaches. Pathway and network approaches are routinely applied in large-scale data analysis for cancer and other complicated diseases. ReactomeFIViz is a Cytoscape app, providing features for researchers to perform pathway- and network-based data analysis and visualization by leveraging manually curated Reactome pathways and highly reliable Reactome functional interaction network. To facilitate adoption of this app in bioinformatics software pipeline and workflow development, we develop a CyREST API for ReactomeFIViz by exposing some major features in the app. We describe a use case to demonstrate the use of this API in a Python-based notebook, and believe the new API will provide the community a convenient and powerful tool to perform pathway- and network-based data analysis and visualization using our app in an automatic way.", "keywords": [ "ReactomeFIViz", "CyREST", "Cytoscape", "Reactome", "Pathway and network" ], "content": "Introduction\n\nPathway- and network-based computational approaches are now routinely used in large-scale data analysis to uncover hidden patterns that are otherwise impossible to discover. These approaches project significant genes, proteins, metabolites, and other kinds of biological entities collected from pre-analysis onto pathways and networks, knowledge produced by many years’ experimental studies. Cytoscape1 is the most popular biological network visualization and analysis platform, widely used in the research community to perform pathway and network analysis and visualization. The release of the CyREST app2 enables Cytoscape as an integrative and indisposable tool to build automatic software pipeline and workflow in programming languages widely used by the bioinformatics and computational biology community, including Python and R, via a RESTful API. The standalone Java-based Cytoscape application thereby functions as a microservice servlet exposing the major features of Cytoscape.\n\nReactome3 is the most comprehensive open source biological pathway knowledgebase, widely used in the research community, with its web site accessed by roughly 60,000 unique IP addresses per month. To perform genome-scale network-based data analysis and visualization, we have also constructed a highly reliable Reactome functional interaction (FI) network by extracting FIs from manually curated pathways from Reactome and other popular large-scale pathway databases and predicting FIs based on a machine learning approach4. Based on this FI network and the high quality Reactome pathways, we have developed a Cytoscape app, called “ReactomeFIViz”5, which is one of most popular Cytoscape apps, downloaded over 30,000 times since it was released in September, 2013 into Cytoscape app store.\n\nReactomeFIViz provides a suite of features to help users to perform pathway- and network-based data analysis and visualization for cancer and other complicated diseases. Users can construct a FI subnetwork based on a set of genes, perform network clustering, annotate found network modules using Reactome pathways and Gene Ontology terms, and perform survival analysis if clinical data is available to search for gene signatures related to patient overall survival. Users can also explore Reactome pathways directly inside Cytoscape, perform pathway enrichment analysis for a set of genes, and conduct pathway modeling using multiple types of omics data based on factor graphs converted automatically from Reactome pathways. Recently we added new features for users to visualize FDA approved cancer drugs and their targets interactions in the contexts of Reactome pathways and the FI network, and perform fuzzy logic based modeling to study the effects of drug application on the pathway activities (see ReactomeFIViz wiki page).\n\nTo facilitate third-party software tool developers to integrate the powerful network and pathway analysis features provided in ReactomeFIViz, we implemented a new CyREST API. The current version of this API is focused on FI network construction and Reactome pathway enrichment analysis for a set of genes.\n\n\nMethods\n\nTo develop the CyREST API for ReactomeFIViz, we followed the recommended procedures described in Cytoscape wiki on adding Automation to existing apps. To handle the complex data models used in ReactomeFIViz, we chose the Functions over Commands approach by adding JAX-RS resource onto existing ReactomeFIViz code base. In brief, we added two new Java packages, org.reactome.cytoscape.rest and org.reactome.cytoscape.rest.tasks, and refactored original tasks into ObservableTask. All refactored ObservableTasks are grouped into package org.reactome.cytoscape.rest.tasks, and their execution is managed by a SyncrhonousTaskManager object and monitored by their respective TaskObserver objects.\n\nThe ReactomeFIViz CyREST API is specified in a Java interface, ReactomeFIVizResource, and documented using Swagger UI as Java annotations for methods defined in the interface. The implementation of ReactomeFIVizResource is provided in class ReactomeFIVizResourceImp. Both the interface and the implementation are placed in package org.reactome.cytoscape.rest.\n\nThe ReactomeFIViz CyREST API is powered up by the CyREST app using its embedded light-weight Grizzly HTTP server. CyREST delegates all RESTful API calls to ReactomeFIViz, which then calls the ReactomeFIViz RESTful server via its RESTful API. The ReactomeFIViz server fetches the Reactome content from databases hosted in a MySQL database engine via a Hibernate API and the in-house built Reactome Java API (Figure 1).\n\n\nOperation\n\nCurrently, the ReactomeFIViz CyREST API provides 8 methods (Table 1). These methods allow third-party workflow and pipeline developers to construct a FI sub-network based on a set of genes listed in a variety of file formats, annotate displayed network using collected pathways, GO biological process, molecular function, or cellular component terms, perform network cluster and then annotate network modules. These methods also allow them to perform Reactome pathway enrichment analysis for a set of genes and then export pathway diagrams. The CyREST API document for ReactomeFIViz, which is accessed via menu Help/Automation/CyREST API, provides detailed description about all these resources.\n\nNotes: 1. GO BP: Gene Ontology biological process; 2. MF: Molecular function; 3. CC: Cellular component.\n\n\nResults\n\nReactomeFIViz CyREST API provides a set of URL-based language-neutral functions, which accept parameters and return results in the JSON format. As with any other CyREST API, it can be easily integrated into Python, R, or any other programming language as long as it supports HTTP-based function calling. Here we describe a use case based on The Jupyter Notebook to showcase the usage of this API in a workflow development.\n\nPrevious study6 has shown the genomic similarity between high-grade serous ovarian tumors and basal-like breast tumors based on multiple omics data types, including copy number variants (CNVs), somatic mutations, and mRNA gene expression. To demonstrate use of the ReactomeFIViz CyREST API, we perform a network- and pathway-based comparison analysis between genes having somatic mutations in TCGA ovarian cancer and breast cancer. Our analysis is focused on showing the utility of our API. Therefore, we use all TCGA breast cancer samples without subtyping to simplify our workflow.\n\nFigure 2 shows the workflow of this use case. The TCGA BRCA (breast invasive carcinoma) and OV (ovarian serous cystadenocarcinoma) mutation data was downloaded from the Broad firehose web site in the mutation annotation file (MAF) format using its RESTful API, and stored in two local files, one for each cancer type. The MAF was then loaded into Cytoscape via the CyREST API to construct a FI sub-network after choosing a sample cutoff to select genes forming a network composed of about 500 genes. The FI-network was then subject to network clustering analysis. Two sets of network modules from network clustering were compared to find modules shared and not shared between these two cancers. Pathway enrichment analysis was performed to collect pathways not shared between them. These results suggest common and cancer-specific network and pathway patterns, facilitating researchers to understand shared and specific oncogenesis mechanisms in these two cancers.\n\nWe performed a network comparison study for the two FI subnetworks constructed separately for TCGA BRCA and OV mutated genes. Network clustering yielded 23 and 38 modules with the TCGA BRCA and OV FI subnetwork, respectively. Overlapping analysis for modules having no less than 3 genes showed 16 out of 18 BRCA modules significantly overlapped with 15 out of 22 OV modules (FDR < 0.01. Results not shown here. See the output in the notebook for details), implying that almost all of BRCA modules can be found in the OV subnetwork and some of BRCA modules are shared with more than one OV module. For example, BRCA Module 1 is significantly overlapped with OV Modules 2 and 3.\n\nFor the pathway comparison study, we focused on pathways enriched for network modules not shared between BRCA and OV, searching for possible different oncogenic mechanisms for these two cancers. The module overlapping analysis showed that BRCA modules 13 and 17, which contains 6 and 4 genes, respectively, are not significantly shared with any OV module. Pathway annotation for these two modules revealed genes in these two modules are significantly enriched for some signaling pathways, including DAG and IP3 signaling, Signaling by GPCR, and Glucagon signaling in metabolic regulation. There are 7 modules in the OV subnetwork that are not significantly shared with the BRCA subnetwork. Pathway enrichment analysis showed that genes in these modules are significantly involved in Ion channel transport, O-linked glycosylation, C-type lectin receptors, and several others. For detailed results, see the output from the notebook.\n\nTo visualize mutated genes in the context of Reactome pathways, the notebook also generated two pathway diagrams, one for each cancer, and saved into the working directory as PDF files. Entities in pathway diagrams composed of mutated genes are highlighted in purple.\n\n\nDiscussion\n\nReactome provides a large set of high-quality manually curated pathways. The Reactome FI network provides a genome-scale highly reliable functional interaction network covering over 60% of total human genes. The ReactomeFIViz CyREST API delivers language neutral REST-based functions for third-party software developers to leverage high-valued resources provided by the Reactome project in their own software tools.\n\nThe current set of functions implemented in this version of ReactomeFIViz CyREST API focuses on some major features implemented in ReactomeFIVz, related to FI network construction, clustering, and Reactome pathway enrichment analysis. As shown in the above use case, it is very easy to integrate with other CyREST APIs and integrated into a Python or R programming language environment to perform Reactome-related pathway and network analysis and visualization.\n\nWe will expose other ReactomeFIViz features in the CyREST API, including gene expression data analysis, network module-based survival analysis, pathway modeling based on Boolean network and probabilistic graph model, and cancer drug visualization and simulation. We will also develop a Python package for easy third-party tool integration.\n\n\nSoftware availability\n\nHome page for user guide: https://reactome.org/tools/reactome-fiviz\n\nCytoscape app store: http://apps.cytoscape.org/apps/reactomefiplugin\n\nLatest source code: https://github.com/reactome-fi/CytoscapePlugIn/tree/path-x\n\nUse case Jupyter notebook: https://github.com/reactome-fi/workflows\n\nSource code at the time of publication: https://github.com/reactome-fi/CytoscapePlugIn/releases/tag/f1000_auto_paper for ReactomeFIViz, https://github.com/reactome-fi/workflows/releases/tag/f1000_auto_paper for the Python use case notebook\n\nArchived source code at the time of publication: http://doi.org/10.5281/zenodo.12264337 for ReactomeFIViz, http://doi.org/10.5281/zenodo.12264308 for the Python use case notebook\n\nLicense: The Creative Commons Attribution 3.0 Unported License (http://www.reactome.org/?page_id=362).\n\n\nData availability\n\nOutput from the network comparison are available in the Python use case notebook http://doi.org/10.5281/zenodo.12264308", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project is supported by a NIH grant (5U41HG003751).\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 would like to thank the whole Reactome team for providing the foundation for ReactomeFIViz.\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): 2498–504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOno K, Muetze T, Kolishovski G, et al.: CyREST: Turbocharging Cytoscape Access for External Tools via a RESTful API [version 1; referees: 2 approved]. F1000Res. 2015; 4: 478. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFabregat A, Jupe S, Matthews L, et al.: The Reactome Pathway Knowledgebase. Nucleic Acids Res. 2018; 46(D1): D649–D655. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu G, Feng X, Stein L: A human functional protein interaction network and its application to cancer data analysis. Genome Biol. 2010; 11(5): R53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu G, Dawson E, Duong A, et al.: ReactomeFIViz: a Cytoscape app for pathway and network-based data analysis [version 2; referees: 2 approved]. F1000Res. 2014; 3: 146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCancer Genome Atlas Network: Comprehensive molecular portraits of human breast tumours. Nature. 2012; 490(7418): 61–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu G: reactome-fi/CytoscapePlugIn: F1000Research ReactomeFIViz Automation (Version f1000_auto_paper). Zenodo. 2018. Data Source\n\nLoney F, Wu G: reactome-fi/workflows: F1000Research ReactomeFIViz Automation (Version f1000_auto_paper). Zenodo. 2018. Data Source" }	[ { "id": "33745", "date": "10 May 2018", "name": "Marina Piccirillo", "expertise": [ "Reviewer Expertise Bioinformatics and 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 authors describe the development of CyREST API for ReactomeFIViz, a Cytoscape app, which exposes some Reactome functions as REST APIs for external software components, to process pathway and network data in automatic and reproducible workflows built using almost any programming languages. The source code provided for ReactomeFiViz and for the Python use case notebook, make the use of CyREST API easier and simpler to understand.\nThe manuscript is well-organized and the described app will be highly valuable for users working with big data related to enrichment analysis and visualization of networks.\nI have only some minor comments:\n\nIn the Introduction section (pag.2): the sentence “These approaches project significant genes, proteins, metabolites, and other kinds of biological entities collected from pre-analysis onto pathways and networks, knowledge produced by many years’ experimental studies.”, is not quite understandable, I think it should be rephrased.\n\nThe Methods section (pag.2) is clearly explained and sufficient details are provided to allow replication of the method development and its use by others.\n\nThe section Operation (pag.2) I think is unnecessary, the authors can add it as the subsection of Methods because here they are explaining the methods available in the CyREST API developed.\n\nIn the Analysis results subsection (pag.3): I think it's possible to add a figure (such as a Venn Diagram) to better explain the results obtained.\n\nIn the same subsection (Analysis results: pag.3) more details should be provided about the results. (For example:\n-\n\nin BRCA modules 13 and 17 are there overlapping genes? If yes, which are their names?\n-\n\nWhat is the way, in which the enriched signaling pathways are involved in BRCA and/or OV carcinoma?)\n\nMore references to supporting literature must be provided in the manuscript in order to further substantiate the claims.\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": "3657", "date": "23 May 2018", "name": "Guanming Wu", "role": "Author Response", "response": "Dear Dr. Piccirillo, Thanks a lot for reviewing our paper and your comments. We have made changes to address some of your comments. Please see details below: 1). In the Introduction section (pag.2): the sentence “These approaches project significant genes, proteins, metabolites, and other kinds of biological entities collected from pre-analysis onto pathways and networks, knowledge produced by many years’ experimental studies.”, is not quite understandable, I think it should be rephrased. We have rephrased this sentence and hope the meaning is clear now. 2). The Methods section (pag.2) is clearly explained and sufficient details are provided to allow replication of the method development and its use by others. Thanks! 3). The section Operation (pag.2) I think is unnecessary, the authors can add it as the subsection of Methods because here they are explaining the methods available in the CyREST API developed. This section is suggested by the article template provided by the journal editor. 4). In the Analysis results subsection (pag.3): I think it's possible to add a figure (such as a Venn Diagram) to better explain the results obtained. Thanks for the suggestion. However, we found that it is difficult to show this type of overlapping result in a Venn diagram. We hope that our text description is clear. 5). In the same subsection (Analysis results: pag.3) more details should be provided about the results. (For example: - in BRCA modules 13 and 17 are there overlapping genes? If yes, which are their names? We have added another sentence to address your question in the second paragraph of the Analysis Result section. - What is the way, in which the enriched signaling pathways are involved in BRCA and/or OV carcinoma?) This is a great question. However, this short software article is written to showcase how to use the new ReactomeFIViz CyREST API. We have refrained ourselves to discuss the biological implications of our analysis results. However, the exported pathway diagrams may answer questions like this, which will be addressed in our future projects. 6). More references to supporting literature must be provided in the manuscript in order to further substantiate the claims. Thanks for the comment. Along with listed references, we have also linked to external resources using hyper-links in the main text. We feel that we have cited enough supporting references for this on-line short software article. Best regards, Guanming Wu, Ph.D." } ] }, { "id": "33666", "date": "11 May 2018", "name": "John H. Morris", "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 have described their extensions to the popular ReactomeFIViz Cytoscape App to support automation via CyREST. They do an excellent job providing the motivation for their extension. The manuscript was clear and well-written, and I believe that the new REST API's will be quite useful.\nI do have a problem with the manuscript, however, in that in the Workflow section, they state:\n\"The MAF was then loaded into Cytoscape via the CyREST API to construct a FI sub-network after choosing a sample cutoff to select genes forming a network composed of about 500 genes. The FI-network was then subject to network clustering analysis. Two sets of network modules from network clustering were compared to find modules shared and not shared between these two cancers. Pathway enrichment analysis was performed to collect pathways not shared between them.\"\nMy problem with this is that they don't provide us with enough detail on the actual CyREST calls and parameters they used to allow us to understand how to reproduce it. Which part of their analysis was done in Python vs. which was done in Cytoscape through the GUI. It is true that all of my answers exist in the Jupyter notebook they provided, but that's a lot of work to figure out that they used the \"buildFISubNetwork\" method with the \"MAF\" format the fiVersion \"2016\". My suggestion is to add at least enough information either in the figure or the text to help others understand the REST flow for their App without referring to the notebook.\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": "3656", "date": "23 May 2018", "name": "Guanming Wu", "role": "Author Response", "response": "Dear Dr. Morris, Thanks a lot for reviewing our paper and your comment. In version 2 of our paper, we have added a new table listing detailed information about actions performed in the use case workflow (Table 2), and added new words in the main text (the second paragraph in the Workflow section) to help readers understand how to reproduce the workflow without going to the Jupyter notebook. We hope our revisions have addressed your concern. Best regards, Guanming Wu, Ph.D." } ] } ]	1	https://f1000research.com/articles/7-531
https://f1000research.com/articles/7-619/v1	21 May 18	{ "type": "Clinical Practice Article", "title": "A closed system irrigation & drainage technique for surgical evacuation of chronic subdural haematomas", "authors": [ "Haider Kareem", "Hadie Adams", "Hadie Adams" ], "abstract": "Background: Chronic subdural haematoma (CSDH), is a common neurosurgical disorder that is associated with morbidity and mortality affecting the ageing population. The aim is to present the treatment experience of CSDH patients treated with a technique that combines the classical single burr-hole irrigation and the continuous closed system drainage: The closed system irrigation & drainage (CSID) technique. Methods: The cases undergoing CSDH evacuation with the CSID method were captured over a 4-year period at a tertiary neurosurgical centre. The authors describe the performance of this methods with respect to post-operative clinical and radiological features, including recurrence rates, complications, and length of stay. Results: A total of 36 cases undergoing 42 CSID procedures (30 unilateral and 6 bilateral CSDHs) were performed, in cases ranging between 55-95 years old (median age 79 years). The rate of recurrence or significant ruminant blood in the subdural space on post-operative imaging was 11% (n=4). No cases of pneumocephalus were observed in this series (n=0). The mean (SD) skin-to-skin time for this procedure was 13.4 (4.4) minutes, with a mean (SD) length of stay of 4 (1.9) days. Conclusion: We conclude that the one burr-hole closed system irrigation and drainage technique with a sub-periosteal drain seems to be a simple, effective and safe procedure for treatment of CSDH. It’s well tolerated under local anaesthesia for patients with high co-morbidities and these preliminary results indicated it may potentially be a better option for treatment of CSDH with a lower rate of post-operative complications.", "keywords": [ "Chronic Subdural Heamatoma", "Sub-periosteal drain", "Close System Irrigation and Drainage (CSID)", "Single Burr-Hole" ], "content": "Introduction\n\nChronic subdural haematoma (CSDH), is a common neurosurgical disorder that is associated with high morbidity and mortality affecting the ageing population. The projected ageing of the population, and the steady increase in the use of anti-platelets and anticoagulants in modern preventive medicine, will expectedly lead to a rising incidence of anticoagulant-related complications, including CSDH1. Its incidence is about (5 per 100 000 per year) in the general population, but is higher for those aged 70 years and older (58 per 100 000 per year)2. The three most common described surgical techniques for subdural hematoma evacuation are: craniotomy, burr hole trephination, and twist-drill trephination3. A randomised controlled trial by Santarius et al. in 2009 concluded that placement of a subdural drain after burr-hole evacuation of CSDH was associated with reduced recurrence rate. Recent studies reported that there are no significant differences in post-operative complication and mortality rate between the type of drain used, whether subperiosteal or subdural drain4–6. In addition to the risk of recurrence, one of the common immediate post-operative complications is pneumocephalus, CSDHs are commonly associated with cerebral atrophy and the associated increase in potential dead space in the subdural area. This factor, in addition to head positioning of the patient’s head during surgery in a neutral supine or slightly tilt, can lead to air collecting at the frontal pole of the subdural space. This can range from simple benign pneumocephalus to tension pneumocephalus with significant morbidity and mortality7,8.\n\nWe present a prospectively collected case series to present a simple surgical technique in the treatment of a common neurosurgical condition, patients presenting with a CSDH requiring surgical evacuation. This technique combines the classical burr-hole irrigation technique and the continuous closed system drainage technique by irrigating in a closed system with continues drainage: the closed system irrigation & drainage (CSID) technique. The modified irrigation technique of this approach is to minimise the operative time, risk of recurrence, and the risk of post-operative pneumocephalus. This study reflects a single surgeon’s experience in the treatment of this condition with cases treated and followed-up over a period of 4 years. We believe that this procedure has proven to be simple, effective, with a relatively short operative time and a low risk of intra- and post-operative complications. Given that the procedure can effectively be done with a single burr hole trephination, we believe that this procedure is easily tolerated under local anaesthesia which is suitable in the treatment of elderly patients with high comorbidities and those unlikely to tolerate general anaesthesia which is common amongst the population presenting with this neurosurgical disorder.\n\n\nMethods\n\nThis case series involves data collected at pre-intervention and post-intervention within routine clinical practice. The UK National Health Service National Research Ethics Service guidance on such research (National Health Service Health Research Authority, 2011)9 determined that the study did not require ethical appraisal or clearance, as it was an evaluation of routine practice/National Health Service audit. Pictures and video recording of the intraoperative surgical procedure were used, alongside pre- and post-operative CT images of the same case for which informed written consent was obtained.\n\nIn this prospective cohort, we aimed to present the data of 36 consecutive patients who underwent surgery for symptomatic chronic subdural hematomas from 2014 to 2018 by a single surgeon (H.K.) at the Imperial College London NHS Trust (locations: Charing Cross Hospital and St Mary’s Hospital). Data from a total of 36 patients receiving 42 procedures are presented in this series. General patient data, including age, sex, secondary diagnosis, drug history, and other risk factors, as well as preoperative symptoms, Glasgow Coma Scale (GCS) at admission, and findings of preoperative Computed Tomography (CT) studies were collected. The side of the haematoma (right, left or bilateral), postoperative symptoms, findings of postoperative CT scan, complications, the rate of recurrence or remnant hematoma were also captured. All patients underwent single-hole trepanation, intraoperative Jacques catheter irrigation, and placement of subperiosteal closed drainage system as the first surgical treatment.\n\n\nOperative technique\n\nPrior to surgery, all patients received full clinical assessment and workup, including reversal of anticoagulation if applicable and consented/assented according to the local protocols for the procedure. One dose of prophylactic antibiotic is usually given at the time of skin incision. The patient is positioned supine or slight lateral position by insertion of gel pads or sandbags under the shoulder and pelvis ipsilateral to the side of haematoma (Figure 1A & 1B). In case of bilateral CSDH, each side is done separately. The head of the patients is tilted to the opposite side. After skin preparation, marking the site of trepanation is at the maximum thickness of the haematoma, which should be the most superior part of the head and parallel to the floor, to avoid air entrapment in the subdural space. A mixed long and short-acting local anaesthetic agent is injected into the incision site, the drain exits and drain’s stitching point, which is usually generously infiltrated especially in the local anaesthetics cases (Figure 2). A skin incision is made about 5cm long, followed by meticulous haemostasis of the skin edge to prevent tracking of fresh blood from the skin to the subdural space at the later stage of the procedure. Following dissection of the periosteum, a self-retaining retractor is then inserted, we usually make sure that there is no bleeding from the edges of the skin or any other point before retractor inserted. Periosteal elevation and burr hole fenestration of the skull is subsequently undertaken, haemostasis of the bone edge and bipolar diathermy of the dura matter preparing for dural opening (Figure 3). The dura is opened in a cruciate fashion with complete cauterisation of the edges to ensure dural retraction and creating small dural defect equivalent to the osseous fenestration. At this stage, the blood starts draining from the subdural space. We make sure to fenestrate any visible membrane over the surface of the brain until the brain is visible without chasing the membrane beyond the dural defect to avoid bleeding from a remote area of the subdural space. The tip of Jacques catheter is then inserted into the subdural space for few millimetre and size 10 subperiosteal closed gravity drain inserted and tunnelled at least 5–7 cm from the surgical site. At this stage, we make sure that the fenestrated part of the sub-periosteal drain is overlying the osseous and dural defect (Figure 4). The Jacques catheter remains at the anterior end of the surgical wound, we remove the retractor and start repairing the galea with vicryl sutures, followed by skin closure around the Jacques catheter (Figure 5). The subdural collection then washed out with warm Ringer's lactate saline with a 50 mL syringe connected Jacques catheter. Irrigating fluid will run to the subdural space through the tip of the Jacques catheter, circulate through subdural space, irrigating all the blood and air from the subdural space and collected through the sub-periosteal drain. The process of slow irrigation continues until the outcoming irrigation fluid becomes clear. When the procedure is under local anaesthesia, we ask the patient, with anaesthetic help, to move the head from one side to another slowly which help in clearing the air and remaining blood in the subdural space. Once the irrigation fluid is clear, we pulled the Jacques catheter from the wound and suture or staple the remaining small opening in the incision (Figure 6). The sub-periosteal drain is usually kept in for 24–48 hours to collect all the subdural ruminant fluid and blood which is further aided by the brain pulsation. In case of bilateral CSDHs, similar steps are then followed on the other side. A video of the procedure is available as part of the supplementary material (Supplementary Video 1). Post-operative CT scan images of the same patient are demonstrated in Figure 7A and 7B.\n\nA) The patient is positioned slight lateral position by insertion of gel pads or sandbags under the shoulder and pelvis ipsilateral to the side of haematoma. B) Marking of skin incision and drain’s exit point.\n\nPrior to dural opening haemostasis of the bone edge and bipolar diathermy of the dura matter is undertaken.\n\nPre- (A) & post-operative (B) [Day 2] cranial CT-scans demonstrating satisfactory evacuation of the subdural collection and expansion of the brain parenchyma with no pneumocephalus in the subdural space.\n\n\nResults\n\nA total of 36 patients, including 26 (72.2%) males and 10 (27.8) females underwent 42 surgical procedures (30 patients unilateral and 6 patients with bilateral trephination). The age ranges from 55–95 years old with median age 79 years (IQR 14). A total of 20 patients underwent right side surgery, 10 patient left side surgery and 6 patients underwent bilateral trephination. The vast majority, 34 (94.4%) patients had a history of trauma, mainly frequent falls. Two patients had no history of trauma reported by the patients, relatives or carer. Seven (19.4%) patients were on anticoagulation treatment, 13 (36%) patients were on antiplatelet and 4 (11%) patients were on both (Table 1).\n\nThe most common clinical features on admission were hemiparesis (n=26, 72%), decreased level of consciousness with a GCS<13/15 (n=19, 52.7%), headache (n=18, 50%), confusion (n=11,30.5%), dysphasia (n=2, 5.5%) and seizures (n=1,2.7%).\n\nAll patients were re-examined post-operatively with regard to presenting symptoms. Post-operative cranial imaging (CT-scan) was done routinely 24–48 hours after the operation as a baseline investigation and also to exclude remnant haematoma or pneumocephalus (Figure 7A & 7B), CT imaging was also performed on clinical follow-up after 1–3 months if indicated. Postoperative pneumocephalus was excluded in all patients. Symptomatic haematoma recurrence developed in 4 patients (11%), 2 of them underwent re-operation in form of mini-craniotomy with excision of the membranes in multi-layered CSDHs. The other 2 patients were treated conservatively as they were not fit for general anaesthesia. Five patients whose CT scan showed a small residual haematoma, but without any neurological deﬁcit, were followed up with satisfactory long-term resolution of the pre-operative symptoms. Postoperative complications developed in 3 patients, one patient suffered partial motor seizures that were managed successfully with anticonvulsant drugs, the two other patients suffered from pneumonia. The mean (SD) length of stay in the neurosurgical unit up until repatriation to secondary care was 4 (1.9) days (Table 2). Further post-operative events, including complications, are summarised in Table 2.\n\n\nDiscussion\n\nAlthough there is a wide range of opinions about the proper surgical management of this common neurosurgical condition, a scoping literature review shows no significant differences in mortality or complications rates between different surgical techniques, and complication rates of the closed draining system after subdural irrigation seems to be similar to the tradition burr-hole irrigation of the subdural space. In addition to that, there were no obvious differences in recurrence between irrigation and no irrigation. However, using a closed drain system seems to be associated with a reduction of the recurrence rate of subdural collection in compare with burr-hole evacuation only2,10,11. In 2009, Zumofen et al. published the first series of patients treated with placement of a sub-periosteal drain system instead of the widely used subdural drain system after double burr-hole craniotomy for hematoma evacuation. They reported equal-to-superior results compared with previous studies regarding hematoma recurrence, mortality, and serious complications, especially postoperative seizures12.\n\nThere is evidence in the literature suggest that sudden evacuation of chronic subdural hematoma may lead to growth or development of remote (contralateral) subdural, epidural or intra-cerebral haematomas13–17. The possible mechanisms behind this phenomenon include the rapid shift of the cerebral hemisphere following hematoma evacuation, over drainage of the subdural space, and cerebral hyperperfusion syndrome, although the actual pathophysiology behind this process remains unclear16–20. We suggest that the CSID technique will allow for a gradual, and physiological more natural, expansion of the brain during the irrigation and drainage process. Unlike the classical burr-hole irrigation technique, with the CSID method, the subdural collection will be slowly substituted with irrigation fluid in a closed system. Similar to the continuous closed system drainage techniques, the CSID method will also allow continues drainage without the introduction of air in the subdural space or rapid shift, however, it also offers the advantages of thorough irrigation. The cerebral pulsations will allow for a slow expansion of the brain parenchyma and a gentler expulsion of the irrigation fluid and possible remaining blood products in the subdural space through the indolent subperiosteal drain.\n\nIt has been widely described that air in the subdural space can physically block re-expansion of the brain following the evacuation of a CSDH. It has also been demonstrated that pneumocephalus increases the recurrence rate of chronic subdural haematomas, and prolongs the hospital stay and healing time21. In the present cohort, no cases of (symptomatic) pneumocephalus were observed, given that post-operative symptomatic pneumocephalus was reported as relatively common complication in the literature, with 8% of the cases undergoing burr-hole irrigation procedures are followed by tension pneumocephalus22–24.\n\nThe rate of recurrence or significant ruminant blood in the subdural space on post-operative imaging was 11% (4 patients). Surgical intervention in the form of a mini-craniotomy was needed in 2 patients. Intra-operatively these cases showed multi-layered membrane adhesions to the surface of the brain. The other 2 patients with symptomatic recurrence were treated conservatively as they were not appropriate candidates for general anaesthesia to undergo a mini-craniotomy due to other significant comorbidities. We didn’t think that they would benefit from revision burr hole evacuation under local anaesthesia due to thick membrane obvious in their CT scan and failure of the first attempt to achieve the target. Reviewing the available literature, re-intervention rates after burr hole surgery with subdural drains vary between 8.3% and 30%3,25–29, with our results falling within this range.\n\nPost-operative infection is an uncommon complication after surgical evacuation of CSDH, with postoperative empyema being reported to occur in approximately 2% of patients using burr-hole evacuation with or without subdural drain methods30,31. We didn’t observe any deep tissue infection. One diabetic patient developed a superficial skin infection which responded well to oral antibiotic treatment.\n\nThe main limitation of this study was its small sample size. Despite including cases from a period of 4 years, the sample size of the final cohort was relatively small given it was a single centre and single surgeons experience, which can all introduce bias to our results. Whether this study lacked the adequate sample size to detect a significant difference between the presented cohort and the numbers presented in the literature or whether the results were indeed comparable remains a question. Another limitation is the inability to test risk factors that could have had an impact on recurrence or other post-operative complications. Also, it was difficult to evaluate its performance in patients with bilateral CSDHs because of the small number of patients with bilateral collections.\n\n\nConclusion\n\nOne burr-hole closed irrigation and drainage technique with a sub-periosteal drain seems to be a simple, effective and safe procedure for treatment of CSDH. It’s well tolerated under local anaesthesia for patients with high co-morbidities and with a low rate of post-operative complication. Larger series are needed to directly compare this technique’s long-term performance to other established methods, however, these preliminary results indicated it may potentially be a better option for treatment of CSDH with a lower rate of post-operative complications.\n\n\nData availability\n\nData cannot be shared because the routinely collected patient data used in the study belong to the Imperial College London and Imperial College Healthcare NHS Trust and not the authors. Researchers can apply to access the data through the Trust’s R&D Department at jrco@imperial.ac.uk.", "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 Video 1 - A step-by-step video demonstrating the closed system irrigation & drainage technique for surgical evacuation of chronic subdural haematomas.\n\nClick here to access the data.\n\n\nReferences\n\nMckissock W, Richardson A, Bloom WH: Subdural heamatoma: a review of 389 cases. Lancet. 1960; 275(7139): 4.\n\nSantarius T, Kirkpatrick PJ, Ganesan D, et al.: Use of drains versus no drains after burr-hole evacuation of chronic subdural haematoma: a randomised controlled trial. Lancet. 2009; 374(9695): 1067–73. PubMed Abstract \| Publisher Full Text\n\nWeigel R, Schmiedek P, Krauss JK: Outcome of contemporary surgery for chronic subdural haematoma: evidence based review. J Neurol Neurosurg Psychiatry. 2003; 74(7): 937–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBellut D, Woernle CM, Burkhardt JK, et al.: Subdural drainage versus subperiosteal drainage in burr-hole trepanation for symptomatic chronic subdural hematomas. World Neurosurg. 2012; 77(1): 111–8. PubMed Abstract \| Publisher Full Text\n\nChih AN, Hieng AW, Rahman NA, et al.: Subperiosteal Drainage versus Subdural Drainage in the management of Chronic Subdural Hematoma (A Comparative Study). Malays J Med Sci. 2017; 24(1): 21–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGazzeri R, Galarza M, Neroni M, et al.: Continuous subgaleal suction drainage for the treatment of chronic subdural haematoma. Acta Neurochir (Wien). 2007; 149(5): 487–93; discussion 493. PubMed Abstract \| Publisher Full Text\n\nMiele VJ, Sadrolhefazi A, Bailes JE: Influence of head position on the effectiveness of twist drill craniostomy for chronic subdural hematoma. Surg Neurol. 2005; 63(5): 420–3; discussion 423. PubMed Abstract \| Publisher Full Text\n\nSchirmer CM, Heilman CB, Bhardwaj A: Pneumocephalus: case illustrations and review. Neurocrit Care. 2010; 13(1): 152–8. PubMed Abstract \| Publisher Full Text\n\nAuthority NHSHR: Does my project require review by a Research Ethics Committee?2011. Reference Source\n\nXu C, Chen S, Yuan L, et al.: Burr-hole Irrigation with Closed-system Drainage for the Treatment of Chronic Subdural Hematoma: A Meta-analysis. Neurol Med Chir (Tokyo). 2016; 56(2): 62–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLind CR, Lind CJ, Mee EW: Reduction in the number of repeated operations for the treatment of subacute and chronic subdural hematomas by placement of subdural drains. J Neurosurg. 2003; 99(1): 44–6. PubMed Abstract \| Publisher Full Text\n\nZumofen D, Regli L, Levivier M, et al.: Chronic subdural hematomas treated by burr hole trepanation and a subperiostal drainage system. Neurosurgery. 2009; 64(6): 1116–21; discussion 1121–2. PubMed Abstract \| Publisher Full Text\n\nPanourias IG, Skandalakis PN: Contralateral acute epidural haematoma following evacuation of a chronic subdural haematoma with burr-hole craniostomy and continuous closed system drainage: a rare complication. Clin Neurol Neurosurg. 2006; 108(4): 396–9. PubMed Abstract \| Publisher Full Text\n\nSun HL, Chang CJ, Hsieh CT: Contralateral acute subdural hematoma occurring after evacuation of subdural hematoma with coexistent contralateral subdural hygroma. Neurosciences (Riyadh). 2014; 19(3): 229–32. PubMed Abstract \| Free Full Text\n\nEom KS, Kim TY, Park JT: Contralateral acute interdural haematoma occurring after burr hole drainage of chronic subdural haematoma. Br J Neurosurg. 2009; 23(2): 213–5. PubMed Abstract \| Publisher Full Text\n\nCohen-Gadol AA: Remote contralateral intraparenchymal hemorrhage after overdrainage of a chronic subdural hematoma. Int J Surg Case Rep. 2013; 4(10): 834–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim CH, Song GS, Kim YH, et al.: Remote Hemorrhage after Burr Hole Drainage of Chronic Subdural Hematoma. Korean J Neurotrauma. 2017; 13(2): 144–148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGürbüz MS, Karaarslan N, Gök S, et al.: Remote Cerebellar Haemorrhage after Burr Hole Drainage of Chronic Subdural Haematoma: A Case Report. J Clin Diagn Res. 2016; 10(5): PD01–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKobayashi S, Mutoh T, Ishikaw T, et al.: [Remote cerebellar hemorrhage after single burr hole drainage of chronic subdural hematoma of the elderly]. No Shinkei Geka. 2011; 39(8): 755–61. PubMed Abstract \| Publisher Full Text\n\nWang G, Yu J: Remote acute subarachnoid hemorrhage after drainage of chronic subdural hematoma: A case report and review of the literature. Int J Surg Case Rep. 2018; 44: 202–206. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYou CG, Zheng XS: Postoperative pneumocephalus increases the recurrence rate of chronic subdural hematoma. Clin Neurol Neurosurg. 2018; 166: 56–60. PubMed Abstract \| Publisher Full Text\n\nSharma BS, Tewari MK, Khosla VK, et al.: Tension pneumocephalus following evacuation of chronic subdural haematoma. Br J Neurosurg. 1989; 3(3): 381–7. PubMed Abstract \| Publisher Full Text\n\nMori K, Maeda M: Surgical treatment of chronic subdural hematoma in 500 consecutive cases: clinical characteristics, surgical outcome, complications, and recurrence rate. Neurol Med Chir (Tokyo). 2001; 41(8): 371–81. PubMed Abstract \| Publisher Full Text\n\nKravtchouk AD, Likhterman LB, Potapov AA, et al.: Postoperative complications of chronic subdural hematomas: prevention and treatment. Neurosurg Clin N Am. 2000; 11(3): 547–52. PubMed Abstract\n\nWakai S, Hashimoto K, Watanabe N, et al.: Efficacy of closed-system drainage in treating chronic subdural hematoma: a prospective comparative study. Neurosurgery. 1990; 26(5): 771–3. PubMed Abstract \| Publisher Full Text\n\nMatsumoto K, Akagi K, Abekura M, et al.: Recurrence factors for chronic subdural hematomas after burr-hole craniostomy and closed system drainage. Neurol Res. 1999; 21(3): 277–80. PubMed Abstract \| Publisher Full Text\n\nKotwica Z, Brzeziński J: Chronic subdural haematoma treated by burr holes and closed system drainage: personal experience in 131 patients. Br J Neurosurg. 1991; 5(5): 461–5. PubMed Abstract \| Publisher Full Text\n\nErnestus RI, Beldzinski P, Lanfermann H, et al.: Chronic subdural hematoma: surgical treatment and outcome in 104 patients. Surg Neurol. 1997; 48(3): 220–5. PubMed Abstract \| Publisher Full Text\n\nEl-Kadi H, Miele VJ, Kaufman HH: Prognosis of chronic subdural hematomas. Neurosurg Clin N Am. 2000; 11(3): 553–67. PubMed Abstract\n\nRohde V, Graf G, Hassler W: Complications of burr-hole craniostomy and closed-system drainage for chronic subdural hematomas: a retrospective analysis of 376 patients. Neurosurg Rev. 2002; 25(1–2): 89–94. PubMed Abstract \| Publisher Full Text\n\nPencalet P: [Complications of chronic subdural hematoma in the adult]. Neurochirurgie. 2001; 47(5): 491–4. PubMed Abstract" }	[ { "id": "34256", "date": "23 May 2018", "name": "Matthew A. Kirkman", "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 prospective study of 36 consecutive patients with chronic subdural haematoma presenting to a single tertiary UK neurosurgical centre over a 4-year period. The authors evaluated use of the so-called closed system irrigation and drainage (CSID) technique, finding no patients developed pneumocephalus, although recurrence or significant residual blood was seen in 11%. Skin-to-skin time was short (mean 13.4 minutes), and mean length of stay in the neurosurgical unit was 4 days.\n\nThis is an interesting study of a common condition seen in neurosurgical practice, and a useful addition to the literature. The multimedia included add significant value to the paper. The authors discuss the limitations of their study clearly. The data suggests that this technique warrants further study in a larger population. I have a couple of questions for the authors:\n\nDo the authors have any comparative data from patients with CSDH treated at the same centre during the same time period, but with different surgical techniques e.g. standard burr-hole irrigation? It would be interesting to compare (perhaps even statistically) surgical time, complication rates and length of stay between the different approaches. However, I do note that the authors already quote previous literature when discussing the complication rates associated with other surgical approaches to CSDH, which in itself is useful.\n\nTable 1 indicates that one patient had a superficial wound infection in the “Co-morbid conditions” section – can the authors clarify exactly where this patient’s infection was, and if it was the same patient that developed a wound infection after sutrgery (as per Table 2).\n\nIs the background of the cases’ 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 conclusion balanced and justified on the basis of the findings? Yes", "responses": [] }, { "id": "34255", "date": "25 May 2018", "name": "Richard Z. Fu", "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 by authors Kareem H and Adams H presents an interesting prospective case series of patients who received an operation for the diagnosis of chronic subdural haematoma which utilised a novel yet seemingly simple, close system irrigation and drainage technique.\n\nTheir results show a recurrence rate not notably different from that reported in the wider literature. Their result of no pneumocephalus reported in their case series is perhaps the most interesting result. Additionally, they report a short operating time and hospital stay which might represent a potential economic benefit to the health service.\n\nIt would have been of value to note the clinical course of other patients in the unit who underwent a more standard operative intervention during the same time period, and for this to have been compared.\nTheir paper provides a relevant and helpful review of the existing literature. Their conclusions drawn are measured and balanced, with dutiful awareness of the study's limitations.\nTheir results could pave the way for a meaningful larger study to follow, where rates of recurrence, pneumocephalus and the cost benefit ratio of operating time and hospital stay can be further explored.\n\nIs the background of the cases’ 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 conclusion balanced and justified on the basis of the findings? Yes", "responses": [] }, { "id": "34257", "date": "29 May 2018", "name": "Rafid Al-mahfoudh", "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 report on treatment of chronic subdural haematomas with a closed irrigation / drainage system is a good pilot for a larger study to compare the methods used. Allowing a more accurate assessment of any potential benefits compared to the more standard treatment methods - especially for outcomes such as recurrence.\nPlease clarify if the irrigation through the Jacques catheter was performed intraoperatively only or if it was continued postoperatively (if so then give an indication how long this was for).\nOtherwise this is a well written article which I recommend for indexing.\n\nIs the background of the cases’ 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 conclusion balanced and justified on the basis of the findings? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-619
https://f1000research.com/articles/7-618/v1	21 May 18	{ "type": "Research Article", "title": "Peculiarities of the formation and subsequent removal of the circulating immune complexes from the bloodstream during the process of digestion", "authors": [ "Sergej B. Landa", "Pavel V. Korabliov", "Elena V. Semenova", "Michael V. Filatov", "Sergej B. Landa", "Pavel V. Korabliov", "Elena V. Semenova" ], "abstract": "Background: Large protein aggregates, known as circulating immune complexes (CICs), are formed in biological fluids as a result of the development of the body's immune response to various provoking factors. The kinetic characteristics of the formation and removal of immune complexes (ICs), their physical parameters, the isotypic composition of immunoglobulins (Igs) and the antigenic component of the CICs may reflect certain aspects of certain pathological and metabolic processes taking place in humans and animals. The aim of this study is to assess the kinetic characteristics of the formation and removal of the CICs that form in blood after eating. We also analyze the changes in the isotypic composition of Igs of ICs that accompany this biological process in rodents and humans. Methods: We identified the CICs, which differed in size and class of Igs, using dynamic light scattering. To remove ICs from the plasma, we used immune-affinity sedimentation. Monoclonal antibodies for the Igs of different isotypes were added to the plasma samples to determine the isotypic composition of the ICs. Results: A large number of ICs were formed in the blood of rats and humans after eating (food CICs). In rats, food ICs are almost immediately filtered in the liver, without circulating in the bloodstream through the body. In humans, the level of food ICs in the blood increases for 3.5 h after ingestion, then within 7–8 h their gradual removal takes place. It was found that in the process of digestion in humans, the isotypic composition of Igs in the CICs changes and becomes more diverse. Conclusions: The molecular–cellular mechanisms of the formation and utilization of food CICs in humans and rodents do not match completely.", "keywords": [ "сirculating immune complexes", "digestion", "dynamic light scattering method", "isotypes of immunoglobulins" ], "content": "Introduction\n\nCirculating immune complexes (CICs) are macromolecular structures formed as a result of the specific interaction of antigens with bivalent or multivalent antibodies in biological body fluid (such as blood, saliva, urine or cerebrospinal fluid). The composition of the CICs is very diverse: CICs can contain a sufficiently large number of different components, including antigens, immunoglobulins (Igs), lipoproteins, proteins of the compliment system, proteins of coagulation, adhesion1,2.\n\nImmune complexes (ICs) are formed due to the development of the body's humoral immune response to various provoking factors and are thought to reflect certain aspects of this process. It is believed that under normal conditions, CICs are rapidly eliminated from the bloodstream; however, with prolonged antigenic effects, their blood levels may rise3. Sedimentation of CICs on the vessel walls and in basal cell membranes stimulates inflammatory processes in these tissues and cells4, leading to serious tissue damage.\n\nThe content of ICs increases in the blood serum and other biological fluids during various systemic disorders, including rheumatological and autoimmune diseases, viral, bacterial and parasitic infections, malignant neoplasms and acute allergic reactions4–14. The levels of CICs change during the process of pathogenesis, as does their structure1,6,9,15–24. Physico-chemical parameters (primarily size) and biological activity of IC depends on the properties of the antigen and the functional characteristics of the antibodies (including isotype, glycosylation and the ability to bind complement)9,25–28. At the same time, information on CIC composition is more useful than information on free antigens and antibodies. Thus, a comprehensive analysis of CICs can serve as a basis for the diagnosis of various pathologies associated with defects in the functioning of the immune system, and could have prognostic value and allows for the monitoring of the effectiveness of the therapy that is used. Along with this, the detection of ICs in biological fluids, estimation of their dimensionality and determination of the isotypic composition of the Igs component in the CICs will contribute to a better understanding of the characteristics of certain metabolic processes taking place in humans and animals.\n\nWe have developed a relatively simple method of determining the CICs, differentiated by the size and class of Igs, which makes it possible to study the structural and functional features of the organization of native ICs29–32. The method is based on the analysis of the contribution of CICs, formed in biological fluids, to the light scattering recorded using a dynamic light scattering (DLS) spectrometer. This is a sensitive and accurate method, characterized by the nature of the measurement, which does not perturb the system that is being studied. Using this technique, it is possible not only to register the CICs that already exist in the blood plasma, but also to study the formation of new ICs upon the addition of exogenous materials and to estimate their distributions according to sizes, from tens to thousands of nanometres. In addition, DLS makes it possible to identify Igs of different classes and isotypes in the composition of circulating ICs without disturbing the structural integrity of the latter and at their minimum concentrations30,32.\n\nIn the present study, we used the DLS method to assess the peculiarities of the formation and subsequent removal of the CICs from the bloodstream in mammals (rats) and humans during digestion.\n\nThe aim of the present study is to assess the kinetic characteristics of the formation and removal of CICs formed in human blood after ingestion (food CICs), and analyse changes in the isotypic composition of Igs of ICs that accompany this biological process. Given that rodents, in particular rats, are often used as laboratory models in the study of digestion processes, we also performed a series of similar experiments in rats using the DLS method.\n\n\nMethods\n\nThe measurements were performed on a PLSS laser correlation spectrometer (INTOX MED LLC, Russia). We used a heterogeneous research scheme. The measurements were carried out in the band of 2,048 or 8,096 Hz. With these measurement parameters, the time taken to study one sample was about 15 min.\n\nThe particle sizes in the study samples were calculated using previously described software products included with the PLSS laser correlation spectrometer (QELS for Windows, Version 3.2, Division of Molecular and Radiation Biophysics, National Research Center “Kurchatov Institute” B.P.Konstantinov St Petersburg Nuclear Physics Institute, Russia)30,33. The measurement result is presented as a histogram of the particle size distribution (histogram of the subfraction composition), in which the abscissa is the scale of dimensions in nanometers, and the contribution to the total scattering of a sample of particles of a given size in percent is plotted along the ordinate axis. In this case, the total scattering of all particles of the sample is taken as 100%. For each sample, measurements were taken at least three times. Statistical processing of the obtained data was carried out using the regularization algorithm33 with the help of QELS 3.2 software, included in the set of the PLSS laser correlation spectrometer.\n\nSee Supplementary File 1 for more information.\n\nIn the experiment, 10 healthy men aged 24–40 years participated voluntarily. All volunteers were chosen from the staff of the Division of Molecular and Radiation Biophysics where the study was performed (National Research Center “Kurchatov Institute” B.P.Konstantinov St Petersburg Nuclear Physics Institute, Russia). All the participants conducted routine daily activities and were not treated with any medical substances for the period of the experiments. Subjects were excluded if they were taking medication during the period of the experiment. Recruitment of exclusively male volunteers was accidental, and it does not bear any significance in this study.\n\nUse of biological material (plasma) was approved by the Ethical Committee of Polenov Russian Scienific Research Institute of Neurosurgery (Saint Petersburg, Russia) (23/H-18 of 23/10/2017). The Institute is authorized to perform research involving human participants according to guidelines from the Ministry of Healthcare of the Russian Federation (Order of the Ministry of Health of the Russian Federation “On the Ethics Committee of the Ministry of Health of the Russian Federation” of 10.07.2015 № 435n) that are in agreement with the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all donors involved to participate in the study. All blood samples were de-identified.\n\nBlood sampling (5 ml) was done before meals and at 1.5, 3.5, 5, 7, 12 and 14 h after eating. First venous blood withdrawal was performed at 8 A.M. from fasted participants. After that, volunteers were provided with hot three-course breakfast (vegetable salad, main course (either meat or fish with garnish), either tea or coffee with a cake). The acquisition of blood plasma was carried out by the standard method34. As an anticoagulant, heparin was added at a final concentration of 50 U/ml.\n\nRemoval of blood cells was carried out by centrifugation at 1,500g for 15 min. The supernatant was collected and for the sedimentation of platelets and decay products of the cell elements, 1 ml was centrifuged for 30 min at 12,500g. The resulting plasma sample was then diluted three times with standard isotonic phosphate buffer (pH 7.2) containing 10 mM EDTA and 1 mM sodium azide.\n\nTo remove ICs from the plasma, we used immune-affinity sedimentation: an excess of protein A, immobilized on sepharose beads (Pasteur Research Institute of Epidemiology and Microbiology, Russia), was added to the blood plasma. Protein A is a protein isolated from the surface of the cell wall of Staphylococcus aureus, it binds the Igs of mammals well, particularly IgG35.\n\nAfter a 10-min incubation at room temperature, the immobilized protein A, together with the Igs bound thereto, were removed from the plasma by centrifugation of 1,000g for 10 min. The supernatant, which is a blood plasma, devoid of Igs (both in free form and in the composition of ICs), was diluted three times with phosphate buffer.\n\nMonoclonal antibodies to human Igs of different isotypes were added to the plasma samples to determine the isotypic composition of the ICs formed during digestion. Adding antibodies to human Igs of different isotypes to the biological fluid results in the specific binding of ICs containing Igs of this isotype, and in the formation of even larger aggregates.\n\nTo the samples of filtered blood plasma (180 µl), prepared according to standard procedure30,33, 20 μl of a solution of monoclonal murine antibodies to human Igs of different isotypes, both in pure form and in various combinations, were added (See section “Antibody details”). In particular, we used antibodies to human IgG1, IgG2, IgG3, IgG4 and IgA. Previously, the antibodies were diluted five times with standard isotonic phosphate buffer (pH 7.2) containing 10 mM EDTA and 1 mM sodium azide.\n\nIn our study, we used only monoclonal murine antibodies (MMA) to human Igs of different isotypes. All antibodies (Test kit “Diagnostic monoclonal antibodies for light and for heavy polypeptide chains of human immunoglobulins” produced according to Specifications 9398-001-11122195-08) were custom-produced for us by Polignost LLC (Saint Petersburg, Russia).\n\nMMA to human IgA (item number: 1H9)36.\n\nAntibodies were raised in the hybridoma strain originated as the result of hybridization of myeloma PX 653A cells with splenocytes from inbred mice SJL/J immunized with the mixtures of purified myeloma proteins IgA of κ and λ types. Antibodies recognize the epitope of the α chain, and they bind effectively both with human IgA1 and IgA2. MMA were purified with affinity chromatography on protein G.\n\nMMA to human IgG1 (item number: 2C11)37.\n\nAntibodies were raised in the hybridoma strain originated as the result of hybridization of myeloma PX 653A cells with splenocytes from inbred mice SJL/J immunized with the mixture of several human myeloma proteins IgG1. Antibodies were purified with affinity chromatography on protein G.\n\nMMA to IgG2 (item number: 3C7).\n\nMMA to IgG3 (item number: 5G12).\n\nMMA to IgG4 (item number: 5C7).\n\nThe procedures of the latter three MMA productions were the same as described above with application of immunogens IgG2, IgG3 and IgG4, respectively.\n\nIn this study, six healthy adult 250–280 g male Wister strain white albino rats (10–11 weeks old) (FGUP PLZH Rappolovo, Russia) were studied. All procedures for rat care and use were conducted in accordance with the National Standard of the Russian Federation GOST R 53434-2009: Principles of good laboratory practice (introduced 01.03.2010 by Federal Agency for Technical Regulation and Metrology, published by Standardinform, Moscow, 2010). The protocol was approved by scientific committee of the Division of Molecular and Radiation Biophysics of National Research Center \"Kurchatov Institute\" B.P.Konstantinov St Petersburg Nuclear Physics Institute (Gatchina, Russia).\n\nAnimals were individually housed in standard polycarbonate cages (width: 206 mm, depth: 365 mm, height: 197 mm) and maintained under standard conditions of temperature (22 #177; 2°C), humidity (55 ± 10%), artificial 12-hour light–dark cycle (lights on at 8:00 A.M., lights off at 8:00 Р.M.).\n\nThe rats were divided into two groups (n=3): those that received complete dietary rations for 3 days (group one) and those that did not receive any food at all (group two). For our tests, animals were allocated to groups in a random manner. The rats in group one were fed on the nutrient extruded and granulated food designed for experimental rodents (LLC) Laboratorkorm, Russia), and they had ad libitum access to it. All animals had ad libitum access to water.\n\nAll efforts were made to ameliorate any suffering of animals. Rats from both groups were anesthetized for the euthanasia with diethyl ether (PJSC “Medkhimprom”, Russia) through the respiratory route by exposing animals to the ether couples for approximately 2 min in an airtight transparent acrylic jar. Dead rats were opened, and their blood was sampled from the Vena portae and from the Vena cava inferior.\n\nBlood was collected into the standard tubes with a clot activator and incubated at 37°C for 60 min, until the clot was completely formed. The clot was separated and the serum was centrifuged at 1,500g for 15 min. The supernatant was collected and again centrifuged for 30 min at 12,000g. The obtained serum sample was diluted three times with isotonic PBS (pH 7.2) containing 10 mM EDTA and 1 mM sodium azide. Each analysed serum sample was divided into two aliquots, one of which served as a control. To the other, 10% (v/v) of protein A, immobilized on sepharose microspheres (20 μl microspheres were added to 180 μl serum), was added.\n\nStatistical processing of the obtained data was carried out using the regularization algorithm33 through application of software QELS 3.2, included in the set of the PLSS laser correlation spectrometer (QELS for Windows, Version 3.2, National Research Center \"Kurchatov Institute\" B.P.Konstantinov St Petersburg Nuclear Physics Institute, Russia).\n\nIn each individual experiment, the error in determining the mean hydrodynamic size of the light-scattering particles in the study samples of biological fluids can be several tenths of a percent (and this value is reproduced when the experiment is repeated on the same sample). The error in determining the mean hydrodynamic size of the light-scattering particles on a series of equally prepared samples is usually higher by an order of magnitude.\n\nTo analyze the dynamic of accumulation of food ICs and their subsequent dissipation in blood serum samples from 10 volunteers, applying DLS, we calculated mean and standard deviation (SD) at several time points for the following parameters of interest: hydrodynamic radius of the scattering particles, their contribution to the light scattering. Mean inter-group difference was calculated with 99% confidence intervals.\n\nThe same calculations were carried out to evaluate isotypic composition of immunoglobulins of human CIC after eating.\n\nComputations were performed using MS Excel 2007 standard statistical functions (Statistical Package Microsoft Office 97 for Windows, Redmond, USA).\n\n\nResults\n\nIn the experiment, two groups of rats participated: the first group of animals received a complete food ration for 3 days and the second was starved. For the analysis by the DLS method, serum from the rat portal vein of the liver and from the inferior vena cava was used.\n\nTo reveal the CICs that formed during the process of digestion, we applied the approach associated with the selective removal of Igs and their complexes from the analysed blood sample using protein A immobilized on sepharose beads, followed by centrifugation31,32. Comparison of the histograms of the subfractional composition of particles of blood serum samples, those processed with protein A and those not processed obtained by the DLS method, allows for the recording of the presence or absence of a fraction of large scattering particles, the hydrodynamic radius (Rh) of which corresponds to the size of ICs.\n\nThe results from analysis of the blood serum from the portal vein of rats that received a full-fledged food ration are shown in Figure 1. It can be seen that in the blood of the animal, there are large protein aggregates, with an Rh of the order of 200–300 nm (Figure 1A), disappearing after treatment of the serum with protein A immobilized on sepharose microspheres (Figure 1B). We have identified these protein aggregates as food CICs.\n\nHistograms of the particle distribution by the size of the blood serum samples from the portal vein of liver of the rat that received a wholesome food ration for 3 days (A and B) and the rat that starved for 3 days (С). The figure presents the results of analyses of the blood serum samples each taken from one most representative rat from each experimental group. (A, С) Samples of native serums. (B). Sample of serum, treated with protein A, immobilized on the agarose microspheres. The x-axis is the hydrodynamic radius of the particles in nm, the y-axis is the contribution to the scattering in %.\n\nThe portal vein in animals is a venous trunk, through which blood from the stomach and intestine passes into the liver. Thus, in the blood of a satiated rat that is coming from the digestive tract to the liver, a substantial amount of CICs are observed (Figure 1A). In the blood serum taken from the portal vein of the rats that were starved for three days, the CICs are practically absent (Figure 1С).\n\nThe results of research of the blood samples from the portal vein of the liver and from the inferior vena cava of the fed and hungry rats also differ substantially; this is evidenced by the data presented in Figure 2.\n\nHistograms of the particle size distribution of the serum samples of hungry rats (A and B) and rats receiving a wholesome food ration (С and D). Blood was taken from the portal vein of the liver (A and С) and the inferior vena cava (B and D). x-axis, the hydrodynamic radius of the particles in nm; y-axis, the contribution to the scattering in %.\n\nThe inferior vena cava collects venous blood from most of the abdominal and pelvic organs, the walls of these cavities and lower limbs. By comparing blood from the portal vein of the liver and inferior vena cava, it is possible to obtain the information about changes in blood composition due to the filtration processes performed by the liver.\n\nComparison of Figure 2A and B shows no changes in the blood serum particle subfractional composition of the rats that starved for 3 days, before and after filtration in the liver. The contribution to the scattering of the fraction of particles matching the ICs in the blood that enters the liver of their gastrointestinal tract (6.1%, Figure 2A) practically matches the analogous indicator in the blood after the passage through the liver (5.8%, Figure 2B) in the starving rats.\n\nContrary to that, the contribution to the scattering of particles with the Rh that matches the ICs in the blood sample, taken from the portal vein of the rats that received a normal food ration is higher by more than an order of a magnitude than the contribution to scattering of particles of the same size in a blood sample taken from the inferior vena cava (73.3%, Figure 2C versus 5.4%, Figure 2D).\n\nThus, the presented results allow us to state that in the process of digestion, a substantial number of ICs that diffuse into the blood are formed in the intestine of rats. These food ICs are completely utilized in the liver.\n\nIn total, 10 healthy men participated in the experiment. Blood sampling was done before meals and at 1.5, 3.5, 5, 7, 12 and 14 h after eating.\n\nWe determined the total contribution to the scattering of the fraction of ICs and the isotypic composition of the Ig component of the food CICs. Analysis of the results showed that the level of ICs in all inspected donors gradually increase in the blood, reaching its maximum at 3.5 h after eating.\n\nAs seen in Figure 3, the base level of the contribution to the scattering of the fraction of particles matching the ICs in the serum was less than 10%. The maximum IC contribution to the scattering was observed after 3.5 h, when it reached almost 80%. Then, the IC fraction contribution of the scattering decreased, reaching the basal level 14 h after eating.\n\nx-axis, time after eating; y-axis, contribution to the scattering of the fraction of food ICs in %.\n\nTable 1 presents the subfractional composition of the particles (the fraction that matches the ICs) of the serum of all the examined donors before eating and at the point of the maximum accumulation of ICs after eating (after 3.5 h). Before meals, there is an insubstantial variation in the size of the recorded ICs and a more substantial difference in the contribution to the scattering of IC fractions in the donors. At 3.5 h after eating, when the level of food complexes in the blood reaches its maximum, the sizes of the complexes for different donors practically match.\n\nAs reported previously, the addition of antibodies to Igs of different isotypes to the blood plasma results in the specific binding of ICs, containing Igs of this isotype, and the formation of even larger protein structures. The appearance of a new fraction of light-scattering particles with a large Rh on the histograms of the subfractional composition of particles, obtained by the DLS method, attests to the presence of this variation of Igs in the analysed ICs. According to the contribution to the scattering of these high-molecular-weight protein aggregates to the total contribution of all ICs, it is possible to determine the proportion of ICs containing Igs of this isotype30,32.\n\nAnalysis of the isotypic composition of the Ig component of the CICs showed that before eating, IgG1 Igs are predominantly present in CICs of all of the examined donors. After 3.5 hours after ingestion, the isotypic composition of Igs in the IC expands (Figure 4) along with the complexes of IgG1, the number increases to 33.1 ± 1.6% (mean ± SD). (Figure 4B), an insignificant number of IgG3 complexes (4.1 ± 0.9%) appears (Figure 4C), and the content of IgA complexes becomes predominant (42.0 ± 0.8%) (Figure 4D).\n\nThe results of the isotypic composition of Igs of the blood ICs of all examined donors are presented in Table 2.\n\nThus, the process of digestion in humans is accompanied by the formation of the CICs, the level of which reaches a maximum value the blood after 3.5 h. Initially, ICs mainly contains the IgG1, then a set of Ig isotypes in the composition of food ICs becomes more diverse. After 3.5 h, a gradual decrease in the amount of food ICs in the blood begins, and 14 h after eating, their concentration drops to the values corresponding to the initial level (before eating).\n\n\nDiscussion\n\nWe recorded a notable fact: numerous macromolecular structures that efficiently scatter light are formed in the plasma after eating. On the basis of the data presented herein, the bulk of high-molecular-weight aggregates of blood plasma, arising de novo during digestion, is formed by Igs of different isotypes and represents ICs (Figure 4). It is obvious that the appearance of ICs in the blood after ingestion should be accompanied by their subsequent removal through cellular macrophage activity, otherwise their permanent accumulation would be observed. We analysed the kinetic characteristics of complexation during digestion in 10 healthy men. All the examined donors had similar temporal characteristics of the appearance, accumulation and removal of food ICs from the bloodstream. The number of newly formed protein aggregates in the peripheral blood increased by more than 5 times fairly quickly, within about 1 h of eating. Later, within several hours, the level of the CICs gradually increased, reaching its maximum after 3.5 h, after which a smooth decrease in the concentration of food ICs begins. After 7-8 h from that moment, characterized by the highest concentration of CICs in the blood, their level returns to the initial state (Figure 3). Thus, the cycle of appearance and removal of food ICs in humans lasts approximately 12 h.\n\n(A) The total contribution of ICs. (B) The contribution of isotype IgG1 complexes. (C) The contribution of isotype IgG3 complexes. (D) The contribution of IgA complexes. (E) The contribution of complexes of IgG1 and IgG3. (F) The contribution of complexes of IgG1 and IgA. x-axis, the hydrodynamic radius of the particles in nm; y-axis, the contribution to the scattering in %.\n\nWe also performed a series of experiments in rats using the DLS method. We compared the composition of the blood (specifically the presence of the CICs) of rats that were kept hungry for 3 days with similar indicators of fed animals. For these purposes, samples of blood from the portal vein and inferior vena cava of the liver of rats were analysed.\n\nBy analysing the blood from the portal vein, we get information about the composition of the blood before it passes through the liver filtration barriers. The results of our studies show that the digestive process in the rat is accompanied by the appearance of a large protein aggregates with an Rh of the order of 200–300 nm, in the blood taken from the portal vein of a satiated animal, which we identified as ICs (Figure 1). That is, in a satiated rat in the blood coming from the digestive tract to the liver, a substantial amount of the CICs are observed (Figure 1A). In the serum of the rats that were not fed for 3 days, taken from the portal vein, the CICs are practically absent (Figure 1C).\n\nThe lower vena cava collects the venous blood from most of the abdominal and pelvic organs, including the blood that has been filtered out by the liver. By comparing the blood from the portal vein of the liver and the inferior vena cava, we can comprehend the changes in the composition of the blood, due to the detoxification carried out by the liver. Virtually no changes in the composition of the blood samples of hungry rats before and after passage through the liver purification filters, were recorded. In this case, the contribution to the scattering of particles with an Rh, corresponding to the ICs, in a blood sample, taken from the portal vein of rats receiving a normal food ration, greatly exceeds the contribution to the scattering of particles of the same size in a blood sample taken from the inferior vena cava (Figure 2C and D). Thus, we can conclude that the ICs formed during the digestion in rats almost immediately enter the liver with the bloodstream, where they are completely utilized.\n\nThe obtained data show that the mechanisms of formation and removal of food ICs in humans and rodents are different. If in rats, food ICs are almost immediately filtered in the liver without circulating in the future with the blood flow through the body, then CIC level in the human blood rises within 3.5 h after eating, after which the blood cleaning of food ICs takes place for a fairly long time (7–8 h).\n\nThe formation of numerous ICs as a result of food intake in the blood plasma is quite unusual. Ideally, during the digestion, the products of complete effective splitting of organic substances should easily leave the body in the form of water-soluble low-molecular-weight fractions, without clogging or overloading the filtering and excretory systems. However, this is not the case in practice: macromolecular structures consisting of food fragments are found in peripheral blood. The free transition of large organic molecules and/or their blocks in unsplit form through the walls of the digestive tract (primarily the intestine) into the bloodstream is called transcytosis38–40.\n\nThe causes and biological relevance of transcytosis during digestion are not clear. However, it is obvious that it induces a local immune response. The data on the growth of CICs in the human blood during the process of digestion presented herein indirectly confirm this. The digestive tract has a number of barrier functions that protect the body from pathogens that enter the human body from the external environment. Nonspecific barrier mechanisms include the antibacterial properties of saliva, bile and pancreatic juice, the proteolytic activity of secrets, intestinal motorics and the characteristic structure of the surface of the intestinal mucosa. This first line of defence against foreign microorganisms and toxic substances is supplemented by a specific immune defence system localized in the digestive tract, which constitutes an important part of the overall multi-component human immune system. The local immune system of the digestive tract provides two main functions: recognition and induction of tolerance to food antigens and a blocking effect on pathogens41. The joint coordinated actions of epithelial cells, immunoreactive mucosal cells, antibodies and synanthropic microbiota form an adaptive immune response42.\n\nOne of the important functions of the mucous membranes is the production of so-called secretory IgA (sIgA), the level of which is much higher in various parts of the gastrointestinal tract than those of other classes of Igs43. sIgA participates in the protection of mucous surfaces from toxins, viruses and bacteria. The polymeric nature of sIgA provides significant advantages in the fight against foreign antigens: sIgA promotes the destruction of pathogens by proteolytic enzymes while remaining intact44.\n\nsIgA molecules are involved in protection against harmful micro-organisms and exogenous agents through a mechanism called immune exclusion45. In addition, as a result of the interaction of sIgA with the cells of Peyer’s patches, the intestinal transepithelial transfer of Igs from the intestinal lumen to the lymphoid tissues takes place in the intestines46. Finally, in the IC, with antigens in the mucous membranes, sIgA causes a systemic reaction accompanied by inducing the production of anti-inflammatory cytokines and limiting the activation of dendritic cells47. In terms of humoral immunity at mucosal surfaces, sIgA appears thus to combine properties of a neutralizing agent (immune exclusion) and of a mucosal immunostimulant inducing effector immune responses in a noninflammatory context favorable to preserve local homeostasis of the gastrointestinal tract48,49.\n\nEvaluation of the isotypic composition of the food ICs, in our opinion, will contribute to a better understanding of the molecular-cellular mechanisms that support the local immune system of the digestive tract. Analysis of the immunoglobulin component of the ICs showed that before eating, IgG1 was the predominant Ig present in the CICs of all examined donors. During the process of digestion, the isotypic composition of immunoglobulins in the CICs expanded (Figure 4): the number of IgG1 complexes is substantially increased (Figure 4B) and a small number of complexes of IgG3 appear (Figure 4C). In this case, the content of IgA complexes becomes predominant (Figure 4D).\n\nThe phenomenon we observed with the help of the DLS method is likely to be a reflection of the features of the metabolic process in a person. This is accompanied by the entry into the blood of relatively large molecular structures, which are not completely digested in the oral cavity and the gastrointestinal path by digestive enzymes into monomers. By binding with the antibodies, they form ICs of considerable size. Basing on the presented results we arrive at the assumption that digestion in humans is accompanied by transcytosis from the intestine to the blood of not only individual molecules of Igs, vitamins, enzymes or large protein aggregates of incompletely digested food50, but also ICs formed in the digestive tract as a result of activation of the local immune system (prevailing share of ICs with IgA in their structure) (Figure 4). Apparently, subsequent elimination of these food ICs from blood stream is performed by mononuclear phagocytes51. Presumably, in this case we are observing an alternative or additional mechanism of digestion that is activated under certain circumstances that are yet unknown.\n\n\nConclusions\n\nThe mechanisms of the formation and removal of food CICs in humans and rodents are different. In humans, the concentration of food ICs in the blood starts rising fast almost instantly after food intake. It reaches the maximum in approximately 3.5 h, being 7 times its initial level. The ICs concentration returns to the initial level after a further 7–8 h through a gradual decrease. In the process of digestion in humans, the isotypic composition of Igs in the food CICs changes and becomes more diverse with time after eating. The food ICs formed during the digestion in rats almost immediately enter the liver with the bloodstream, where they are completely utilized.\n\n\nData availability\n\nDataset 1. The raw light scattering data for the subjects at each time point, and for the starved and fed rats. DOI: 10.5256/f1000research.14406.d20331152.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nSupplementary File 1. Determination of particles size by means of the method of dynamic light scattering.\n\nClick here to access the data.\n\n\nReferences\n\nOhyama K, Kuroda N: Proteomic approaches to profiling the humoral immune response and identifying disease-associated antigens. Biol Pharm Bull. 2012; 35(9): 1409–1412. PubMed Abstract \| Publisher Full Text\n\nOhyama K, Kuroda N: Immune complexome analysis. Adv Clin Chem. 2013; 60: 129–141. 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Reference Source\n\nLebedev AD, Ivanova MA, Lomakin AV, et al.: Heterodyne quasi-elastic light-scattering instrument for biomedical diagnostics. Appl Opt. 1997; 36(30): 7518–7522. PubMed Abstract \| Publisher Full Text\n\nSmoljanitskiy A: Methods of studying the homeostasis system. In: Menshikov VV (ed.) Laboratory methods of research in the clinic. Moskow: Medicine, 1987; 149–168.\n\nFournier B, Klier A: Protein A gene expression is regulated by DNA supercoiling which is modified by the ArlS-ArlR two-component system of Staphylococcus aureus. Microbiology. 2004; 150(Pt 11): 3807–3819. PubMed Abstract \| Publisher Full Text\n\nSamoilovich MP, Pisareva MS, Klimovich BV, et al.: Epitopes of IgA, sIgA and SC molecules detected with monoclonal antibodies (MA). Medical Immunology (Russia). 2005; 7(2–3): 280.\n\nSamoylovich MP, Krutetskaya IYu, Klimovich VB, et al.: Monoclonal antibodies to human IgG subclasses. Development and specificity determination. Immunologiya (Immunology) (Russia). 1998; 3: 27–30.\n\nMehta D, Malik AB: Signaling mechanisms regulating endothelial permeability. Physiol Rev. 2006; 86(1): 279–367. PubMed Abstract \| Publisher Full Text\n\nCai Q, Wang L, Deng G, et al.: Systemic delivery to central nervous system by engineered PLGA nanoparticles. Am J Transl Res. 2016; 8(2): 749–764. PubMed Abstract \| Free Full Text\n\nLundquist P, Artursson P: Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues. Adv Drug Deliv Rev. 2016; 106(Pt B): 256–276. PubMed Abstract \| Publisher Full Text\n\nBerin MC: Mucosal antibodies in the regulation of tolerance and allergy to foods. Semin Immunopathol. 2012; 34(5): 633–642. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOgra PL, Mestecky J, Lamm ME, et al.: Mucosal Immunology. 2nd ed. San Diego: Academic Press, 1999. Reference Source\n\nWoof JM, Mestecky J: Mucosal immunoglobulins. Immunol Rev. 2005; 206: 64–82. PubMed Abstract \| Publisher Full Text\n\nStubbe H, Berdoz J, Kraehenbuhl JP, et al.: Polymeric IgA is superior to monomeric IgA and IgG carrying the same variable domain in preventing Clostridium difficile toxin A damaging of T84 monolayers. J Immunol. 2000; 164(4): 1952–1960. PubMed Abstract \| Publisher Full Text\n\nNagler-Anderson C: Man the barrier! Strategic defences in the intestinal mucosa. Nat Rev Immunol. 2001; 1(1): 59–67. PubMed Abstract \| Publisher Full Text\n\nRobinson JK, Blanchard TG, Levine AD, et al.: A mucosal IgA-mediated excretory immune system in vivo. J Immunol. 2001; 166(6): 3688–3692. PubMed Abstract \| Publisher Full Text\n\nCorthésy B: Roundtrip ticket for secretory IgA: role in mucosal homeostasis? J Immunol. 2007; 178(1): 27–32. PubMed Abstract \| Publisher Full Text\n\nMacpherson AJ, Hunziker L, McCoy K, et al.: IgA responses in the intestinal mucosa against pathogenic and non-pathogenic microorganisms. Microbes Infect. 2001; 3(12): 1021–1035. PubMed Abstract \| Publisher Full Text\n\nWu HY, Weiner HL: Oral tolerance. Immunol Res. 2003; 28(3): 265–284. PubMed Abstract \| Publisher Full Text\n\nTuma P, Hubbard AL: Transcytosis: crossing cellular barriers. Physiol Rev. 2003; 83(3): 871–932. PubMed Abstract \| Publisher Full Text\n\nShmagel KV, Chereshnev VA: Molecular bases of immune complex pathology. Biochemistry (Mosc). 2009; 74(5): 469–79. PubMed Abstract \| Publisher Full Text\n\nLanda SB, Korabliov PV, Semenova EV, et al.: Dataset 1 in: Peculiarities of the formation and subsequent removal of the circulating immune complexes from the bloodstream during the process of digestion. F1000Research. 2018. Data Source" }	[ { "id": "35398", "date": "03 Jul 2018", "name": "Boris A. Margulis", "expertise": [ "Reviewer Expertise molecular 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 study of Landa and others aims to understand how food digestion process affects circulating immune complexes, CIC. Since the amount of immune complexes increases in biological fluids as the result of various disorders, including autoimmune diseases, viral and bacterial infections and malignancies, the appearance and precipitation of the particles need more careful investigation, particularly as applied to processes of normal physiology. The idea of the study was to measure the content of CICs after food digestion, process believed to increase the particles’ number in plasma. The authors employed the method of dynamic light scattering (DLS) to analyze the size of CIC particles, and the accuracy of this method was proved in studies of ICs from rats and humans after food ingestion. To identify Ig participating in CIC assembly Sepharose microspheres covered with Protein A were used, and both methods allowed to estimate the dynamics of CIC accumulation and lowering in blood. Using this combination of techniques the authors identified the fraction of particles with the size of 200-300 nm as food CIC. The authors stated that most of CIC in a process of digestion appear in blood from intestine and then they are quickly and completely utilized by the liver. In contrary, in humans the accumulation and removal of CICs from blood occurs more slowly than in rodents and takes approximately 12 hours. The cause of such delay is explained by authors by an incomplete digestion of larger molecular structures persisting in food. To define the spectra of Igs in CIC the authors used monoclonal antibodies generated in house. The changes in Ig classes during CIC utilization lasting 7-9 hours after ingestion were strong and it was found that instead of initial predominance of IgGs IgA become prevalent at the end of that period. This change may be hypothetically associated with the necessity of maintaining of immune system in activated state after food consumption. The study of Landa et al evokes several questions and one of the latter concerns the experimental approval of dependence of CIC content and composition on meal volume and/or composition. To conclude, the authors made a big work; they implemented a novel technique, DLS, for the analysis of immune complexes and obtained interesting results concerning particularly the long-living CIC in humans resulting from food consumption.\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": [] }, { "id": "35166", "date": "10 Jul 2018", "name": "József Prechl", "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 measure immunoglobulin containing complexes by dynamic light scattering and the relationship between dietary intake and the presence, amount and quality of these complexes. Experiments are carried out using rats and by obtaining blood from human volunteers. The stated aim of the study is the monitoring of quantity and quality of immune complexes following food intake.\nMethodology Protein A binds either poorly or not at all rat immunoglobulins, so it was not a good choice for the experiment. Therefore the fact that complexes with a hydrodynamic radius of 200-300 nm are removed from rat serum is only indirect and weak evidence that these complexes are immune complexes. Even if these complexes contain immunoglobulins the specific interactions between antibody and antigen should be proved in order to be called immune complexes.\nResults Figure 1A and Figure 2C are identical, redundance could be reduced by merging these figures.\nProtein A treatment seems to alter composition not only by removing large complexes but also by increasing the relative contribution to light scattering of 81-109 nm sized complexes. This is not addressed by the authors. Similar complexes are present in the IVC sample (Figure 2D) of fed versus fasted rats (Fig 2B).\nHow was the fraction of particles matching the ICs in human heparinized plasma determined? Figure 3 and Table 1 is based on the identification of this fraction, yet it is not defined under Methods.\nDiscussion The first two paragraphs on page 11 provide a general overview of secreted IgA but the authors do not explain how that is related to their findings; in the present context it is not relevant and could be omitted.\nIn Table 2 it is not clear whether + sign means OR or AND; IgG1+IgG3 seems to refer to complexes containing either IgG1 OR IgG3, while IgG1+IgA seems to refer to double positive (IgG1 AND IgA) complexes. Figure 4B-D-F suggests that complexes contain exclusively either IgG1 or IgA, these isotype specific monoclonals reacting with roughly half-half of the complexes. This is very unusual for immune complexes where different isotypes against the same antigen are usually present at the same time.\nConclusions The authors claim that the molecular and cellular \"mechanisms of the formation and utilization of food immune complexes in humans and rodents do not match completely\". The experimental and methodological approach was different in rodents and humans, therefore such a conclusion cannot be reached. Kinetics was not followed in rats that were fed ad libitum, evidence is only indirect via the use of two sampling sites (vena portae, vena cava inferior) in contrast to temporal monitoring in humans. No cellular measurements were done at all, therefore conclusion on cellular mechanisms cannot be drawn.\nMinor formal comment Throughout the figures cyrillic \"nm\" is used for nanometer, please replace with latin nm.\nThe topic, how ingested food components are recognized by preformed immunoglobulins and how this recognitions affects food uptake and immunological tolerance and metabolism in general, is very timely and interesting. The article has several weak points though that need to be improved for solid and convincing 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? 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "35397", "date": "20 Jul 2018", "name": "Vadim Govorun", "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 'Peculiarities of the formation and subsequent removal of the circulating immune complexes from the bloodstream during the process of digestion' by Sergej B. Landa et al describes the molecular–cellular mechanisms of the formation and utilization of food CICs in humans and rodents. Circulating immune complexes (CICs) are macromolecular structures formed as a result of the specific interaction of antigens with bivalent or multivalent antibodies in biological body fluid. Authors have developed a relatively simple method of determining the CICs, differentiated by the size and class of Igs, which makes it possible to study the structural and functional features of the organization of native ICs. They found the fact numerous macromolecular structures that efficiently scatter light are formed in the plasma after eating and the mechanisms of formation and removal of food ICs in humans and rodents are different. This manuscript provides a new information about special metabolic process in a person, which accompanied by the entry into the blood of relatively large molecular structures, which are not completely digested in the oral cavity and the gastrointestinal path by digestive enzymes into monomers.\n\nI think, this is a very interesting work, and this manuscript is worthy of publication in your journal.\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/7-618
https://f1000research.com/articles/7-616/v1	21 May 18	{ "type": "Research Article", "title": "Antimicrobial peptide LL-37 and recombinant human mannose-binding lectin express distinct age- and pathogen-specific antimicrobial activity in human newborn cord blood in vitro", "authors": [ "Annette Scheid", "Ning Li", "Carleen Jeffers", "Francesco Borriello", "Sweta Joshi", "Al Ozonoff", "Matthew Pettengill", "Ofer Levy", "Ning Li", "Carleen Jeffers", "Francesco Borriello", "Sweta Joshi", "Al Ozonoff", "Matthew Pettengill" ], "abstract": "Background: There is a need to prevent and treat infection in newborns. One approach is administration of antimicrobial proteins and peptides (APPs) such as LL-37, a membrane-active cathelicidin antimicrobial peptide, and mannose-binding lectin (MBL), a pattern-recognition protein that binds to microbial surface polysaccharides resulting in opsonization and complement activation. Low plasma/serum levels of LL-37 and of MBL have been correlated with infection and exogenous administration of these agents may enhance host defense. Methods: The antimicrobial activity of LL-37 (15 µg/ml) or rMBL (0.5, 2 and 10 µg/ml) was tested in hirudin-anticoagulated preterm and term human cord blood (N = 12–14) against Staphylococcus aureus (SA) USA 300 (2x104 CFU/ml), Staphylococcus epidermis (SE) 1457 (2x104 CFU/ml) and Candida albicans (CA) SC5314 (1x104 CFU/ml). After incubation (1, 45, or 180 min), CFUs were enumerated by plating blood onto agar plates. Supernatants were collected for measurement of MBL via ELISA. Results: Preterm cord blood demonstrated impaired endogenous killing capacity against SA and SE compared to term blood. Addition of LL-37 strongly enhanced antimicrobial/antifungal activity vs SA, SE and CA in term blood and SE and CA in preterm blood. By contrast, rMBL showed modest fungistatic activity vs CA in a sub-analysis of term newborns with high basal MBL levels. Baseline MBL levels varied within preterm and term cohorts with no correlation to gestational age. In summary, exogenous LL-37 demonstrated significant antimicrobial activity against SA, SE and CA in term and SE and CA in preterm human blood tested in vitro. rMBL demonstrated modest antifungal activity in term cord blood of individuals with high baseline MBL levels. Conclusions: To the extent that our in vitro results predict the effects of APPs in vivo, development of APPs for prevention and treatment of infection should take into account host age as well as the target pathogen.", "keywords": [ "Newborn", "Preterm", "Cord Blood", "Antimicrobial Activity", "LL-37", "Mannose Binding Lectin" ], "content": "Introduction\n\nNeonatal sepsis is a major contributor to neonatal morbidity and mortality; consequently, efforts to ease the burden of this disease are crucial1. Sepsis reflects an infection-induced systemic inflammatory response syndrome2. Early-onset sepsis (EOS) is most commonly differentiated from late-onset sepsis (LOS) by the onset occurring before or after the first 72 h of life, respectively3. Of note, both the clinical features and pathophysiology of sepsis varies markedly by age, such that adult, pediatric and neonatal sepsis criteria are distinct1. While screening and prophylaxis for Group B Streptococcus has reduced rates of EOS (i.e., within the first 3 days of life), LOS in the preterm infant has increased in frequency as a higher number of premature infants have survived, resulting in invasive procedures and prolonged hospital stays, as well as increased pathogen exposure3,4. LOS considerably lengthens the infant’s hospital stay, and is associated with long-term neurodevelopmental complications and a high risk of mortality1. Risk of LOS is inversely related to birth weight and gestational age (GA); as such, preterm and very low birth weight infants are at a higher risk of infection5. Accordingly, there is a need to reduce and mitigate neonatal LOS.\n\nOne approach to reducing and mitigating LOS in high-risk newborns is the use of immunomodulatory strategies. Among these, a promising area for investigation are antimicrobial proteins and peptides (APPs)6. For example, administration of oral lactoferrin to preterm newborns reduces the risk of sepsis and necrotizing enterocolitis7. In the present study we focused on the potential utility of two APPs with distinct modes of action: (a) the α-helical LL-37 cationic cathelicidin8 is a broad spectrum membrane-active antimicrobial peptide that induces microbial lysis, blocks endotoxin activity, synergizes with other host defense systems,9 and modulates inflammatory responses9,10; and (b) mannose-binding lectin (MBL), a host pattern recognition receptor that recognizes and binds to sugar moieties on the surface of bacteria and fungi, enhances opsonophagocytosis, and forms complexes with MBL-associated serine proteases that trigger complement activation11. Indeed, relatively low plasma LL-37 or MBL concentrations are associated with a higher risk of infection9,12. Deficiencies in LL-37 or MBL levels can be genotypic13,14, such as genetic variants of exon 1 on the human MBL gene (MBL2), or phenotypic such as reduced expression of APPs in preterm plasma15,16. Some premature infants have a distinct immune system, and some may be MBL-deficient, as defined in prior neonatal studies by plasma/serum concentrations <700 ng/ml11,17. Accordingly, it has been hypothesized that the administration of recombinant MBL (rMBL) as a supplement to bolster the neonatal innate immune system could reduce the risk of LOS11. However, to our knowledge, no published studies have examined addition of rMBL to human newborn blood.\n\nTo characterize the activity of LL-37 and rMBL in neonatal blood, we evaluated antimicrobial activity towards three pathogens commonly associated with LOS in newborns: (a) Staphylococcus epidermidis (SE), that accounts for 78% of cases of LOS due to coagulase-negative staphylococci, (b) Staphylococcus aureus (SA), a less common pathogen associated with a high rate of mortality; and Candida albicans (CA) the most common fungal pathogen associated with LOS. We also conducted a sub-analysis with respect to rMBL effects in term cord blood with low baseline levels vs those with high baseline MBL levels. We found that these agents exerted distinct antimicrobial activity that depended on both pathogen and age. Specifically, rMBL demonstrated modest fungistatic activity vs CA in term newborns with high basal MBL levels. By contrast, LL-37 demonstrated substantial antimicrobial activity that was generally greater in term (SA, SE and CA) than in preterm (SE and CA) blood tested in vitro. The antimicrobial activity of rMBL and LL-37 in vitro depends on three factors: the baseline endogenous level of APP, the pathogen identity and the age of the host, informing the translational development of these promising agents.\n\n\nMethods\n\nrMBL, provided by Shire (Lexington, MA), was expressed in HEK293 cells and purified by affinity chromatography on Glucosamine Sepharose 4FF and ion exchange chromatography on Source 30Q and diafiltration (100 kDa) from GE Healthcare Life Sciences (Pittsburg, PA, USA), including a Benzonase DNA removal step from MilliporeSigma (Billerica, MA) or similar and several microfiltration and nanofiltration steps for bioburden and adventitious virus elimination. rMBL was provided frozen, aliquoted at 10× assay concentration, and stored in single-use quantities to minimize freeze-thaw. LL-37 was purchased from AnaSpec, Inc. (Fremont, California); it was purchased in 1 mg vials, re-suspended in 1 ml distilled water and frozen in aliquots (stock concentration 1 mg/ml) at -80°C.\n\nThe anti-infective effect of rMBL and LL-37 was assessed in three pathogens: (a) SE strain 1457, a clinical isolate from a central catheter infection (kindly provided by Dr. Michael Otto, National Institute of Allergy & Infectious Diseases, National Institutes of Health, Rockville, MD), was cultured in trypticase soy broth (TSB), as previously described18; (b) SA strain USA300, a strain of community-associated methicillin-resistant SA (kindly provided by Dr. William Nauseef, University of Iowa; Iowa City, IA) that was cultured in TSB; and (c) CA strain SC531419, (kindly provided by Dr. Julia Koehler, Division of Infectious Diseases, Boston Children’s Hospital, Boston, MA), which was cultured in yeast extract-peptone-dextrose (YPD) broth.\n\nCord blood was obtained from 30 human newborns: 22 term newborns ranging from 37 0/7 to 40 4/7 weeks GA and 8 preterm newborns ranging from 26 1/7 to 36 6/7 weeks GA. Cord blood samples were collected at The Brigham and Women’s Hospital (BWH) and Beth Israel Deaconess Medical Center (BI), both tertiary care centers for newborn delivery and postnatal care. De-identified newborn cord blood was collected immediately after Caesarian section or vaginal delivery of the placenta from a large umbilical vein and was anti-coagulated with pyrogen-free hirudin (Verum Diagnostica GmbH, Munich, Germany). Since the mechanism of action of MBL involves complement activation, we used Hirudin as an anticoagulant which does not impact complement activation. We did not use Heparin or EDTA as coagulants, as Heparin, is known to bind to complement and EDTA may inhibit complement activation. Inclusion criteria were either term or preterm gestational age; and birth via vaginal delivery or caesarian section. Sample collections included both male and female newborns. Exclusion criteria were maternal fever peripartum (>104°F/40°C) or seropositive status for human immunodeficiency virus.\n\nPatient information concerning the collected cord blood samples was collected in a de-identified manner and hence maternal consent was waived by the local institutional review boards at The Brigham and Women’s Hospital (Protocol #:2000P000117/BWH) and the Beth Israel Deaconess Medical Center (Protocol #2011P-000118/BIDMC. The data associated with our study has been provided in an Excel-compatible format.\n\nA total of 10–20 ml of term or preterm cord blood was collected in hirudin vacutainers at room temperature and processed within 4 h of collection. A total of 1 ml hirudinated blood was centrifuged and plasma collected and cryopreserved at -80°C for subsequent evaluation of MBL concentrations via ELISA (Hycult®biotech; Cat. No. HK323-01). Endogenous LL-37 levels were not determined. LL-37 was prepared at 10× assay concentration in 1× saline. A total of 15 µl negative control (saline), rMBL (500 ng/ml, 2000 ng/ml, or 10,000 ng/ml), or LL-37 reagents (1 mg of protein/ml) as well as 15 µl SA strain USA300 (2×104/ml), SE strain 1457 (2×104/ml) or CA (final concentration 1×104 CFU/ml) in saline were added to 120 µl hirudin-anticoagulated preterm and term human cord blood and incubated at 37°C. At 1, 45, and 180 min, 10–20 µl of each replicate was spread on tryptic soy blood agar plates to quantify colony forming units (CFUs). Plate CFUs were counted 16–18 h after assay commencement for SA and SE, or at 48 h after assay commencement for CA using the Accu Count™ 1000, Automated Colony Counter (BioLogics, Inc.). This automated colony counter was carefully calibrated, and the assay designed to ensure colony counts <200 colonies per plate in order to facilitate reliable colony counts. Of note, the cord blood collection volumes obtained permitted incubation with all three pathogens in 20 of the term patient samples, incubation with SA and SE but not CA in one term sample, and incubation with SA only in one term sample.\n\nData were analyzed and graphed using Prism for MacIntosh v. 7.0 (GraphPad Software, Inc.). Tests used for statistical comparisons are indicated in the figure legends. P values <0.05 were considered significant. Statistical analysis was performed via two-way ANOVA with either a Sidak’s post hoc test (Figure 1, Figure 2 panels (B) and (C), Figure 3 panel (C), Figure 4 panel (C) and Figure 5 panel (C) or Dunnett’s post hoc test (Figure 3 panels (A) and (B), Figure 4 panels (A) and (B), and Figure 5 panels (A) and (B). In Figure 2 panel (A), Spearman’s correlation was performed.\n\n\nResults\n\nOverall, bacterial viability decreased over time in our whole-blood assay (Figure 1). In accordance with the known deficiency of antimicrobial mechanisms in preterm infants, preterm cord blood demonstrated significantly lower killing capacity against SE (Figure 1A) or SA (Figure 1B) than term cord blood at 180 min. The viability of CA increased modestly over time in both preterm and term cord blood, with no significant differences observed between age groups (Figure 1C).\n\nPreterm cord blood exhibits a lower killing capacity against S. epidermidis (A) and S. aureus (B) than term cord blood at 180 min. While the trend towards lower killing activity by preterm cord blood was observed for C. albicans (C) at 45 min, the difference did not reach statistical significance. Killing capacity was measured at 1 min, 45 min and 180 min; the inoculum (SA and SE, 2x104 CFU/ml; CA, 1x104/ml) at time-point “0” is plotted at “0.1 min”. CFU counts are expressed in percent of CFUs detected at 1 min. Term, N = 20–22 (N = 20 for CA; N = 21 for SE; N = 22 for SA); preterm, N = 8. Statistical analysis was performed via two-way ANOVA with Sidak’s post hoc test. **p<0.0001. p<0.005. All graphs depict mean and error with SEM.\n\nIndividuals were stratified into high vs low baseline plasma MBL values, using a threshold of 700 ng/ml, in keeping with past studies11. Baseline MBL concentrations within both preterm and term groups varied broadly (Figure 2A). GA and baseline MBL level were not significantly correlated (Spearman r = 0.18, p = 0.33). This suggests that MBL concentrations did not vary by GA, in agreement with the results of other groups20. In our term cohort, exogenous rMBL, when added to high baseline MBL cord blood, showed a modest fungistatic effect against CA when compared with saline treated control high baseline MBL term cord blood at 180 min (Figure 2C). By contrast, exogenous rMBL demonstrated no bactericidal effect against SE in low or high baseline MBL term cord blood (Figure 2B). With respect to SA, there was no significant effect of high-dose rMBL addition to term cord blood with low or high baseline MBL levels (data not shown).\n\n(A) Scattergram of plasma MBL levels as a function of gestational age. Baseline MBL levels that do not correlate with gestational age. (B) No statistical significance in the antimicrobial effect against SE between MBL treatment vs saline in the low or high baseline MBL groups when analyzed separately. (C) Enhanced antifungal effect of exogenous MBL towards CA in term newborns with high baseline MBL levels only. Statistical analysis was performed in panels (B) and (C) via repeated measure two-way ANOVA with Sidak’s post hoc test. *p<0.005. All graphs depict mean and error with SEM. CFU counts was measured at 1 min, 45 min and 180 min, the inoculum (SA and SE, 2x104 CFU/ml; CA, 1x104 CFU/ml) at time-point “0” is plotted at “0.1 min”. A Spearman correlation was performed for panel (A), which demonstrates no correlation between gestational age and baseline MBL levels in our cohort (r=0.1834, p=0.3319). CFU counts in (B) and (C) are expressed in percent of CFUs detected in saline treated control blood obtained from the same individual at 1 min. (A) Baseline MBL levels detected in a total of 30 samples (22 term, 8 preterm); (B) SE: Low baseline MBL N = 6; High baseline MBL N = 15; (C) CA: Low baseline MBL N = 5; High baseline MBL N = 15.\n\nFigure 3 demonstrates the effects of addition of LL-37 as well as rMBL at three different concentrations on the growth of SA in preterm and term cord blood. In preterm cord blood, the addition of neither rMBL nor LL-37 inhibited the growth of SA relative to the saline control (Figure 3A). By contrast, in term cord blood, LL-37 significantly decreased SA growth at 180 min, whereas rMBL did not (Figure 3B). The inhibitory effect of LL-37 on SA growth was more pronounced in term cord blood than in preterm cord blood (Figure 3C).\n\nViability of SA as measured in CFU is plotted on the y-axis relative to the incubation time in panels (A) and (B). (A) Neither LL-37 nor MBL inhibit growth of SA in preterm blood. (B) LL-37 significantly inhibits SA growth in term blood at 180 min. (C) Summary of LL-37 effects on SA growth in term and preterm cord blood at 180 min, demonstrating that the inhibitory effect of LL-37 is more pronounced in term than in preterm cord blood. The inoculum (2×104 CFU/ml) at time-point “0” is plotted at 0.1 min. Term, N = 20–22; preterm, N = 8. p<0.005. Statistical analysis employed two-way ANOVA with Dunnett’s (A, B) or Sidak’s (C) post hoc test. All graphs depict mean and error with SEM.\n\nAs demonstrated in Figure 4, LL-37 demonstrated a pronounced inhibitory effect on SE growth in both preterm (Figure 4A) and term cord blood (Figure 4B) at 45 and 180 min. In term cord blood this effect was evident at 1 min incubation. rMBL showed no bactericidal effect against SE in preterm (Figure 4A) or term (Figure 4B) cord blood. When comparing the bactericidal effect of LL-37 at 180 min in term cord blood to preterm cord blood, the effect in term cord blood was more pronounced (Figure 4C).\n\nViability of SE as measured in CFU is plotted on the y-axis relative to the incubation time in panels (A) and (B). (A) LL-37 but not MBL inhibits growth of SE in preterm blood at 45 min and 180 min. (B) LL-37 significantly inhibits SE growth in term blood at 1 min, 45 min and 180 min. (C) Summary of LL-37 effects on SE growth in term and preterm cord blood at 180 min, demonstrating that the inhibitory effect of LL-37 is stronger in term than in preterm cord blood. The inoculum (2x104 CFU/ml) at time-point “0” is plotted at 0.1 min. Term N = 21–22 (N = 20 for CA; N = 21 for SE; N = 22 for SA); preterm N = 8. Statistical analyses employed two-way ANOVA with Dunnett’s (A, B) or Sidak’s (C) post hoc test. p<0.05, p<0.0005, p<0.0005. All graphs depict mean and error ± SEM.\n\nFigure 5 demonstrates the significant growth inhibitory effect of LL-37 on CA growth in preterm (Figure 5A) and term cord blood (Figure 5B) relative to saline control at all three time points measured. rMBL demonstrated no inhibitory effect on CA growth in preterm or term cord blood. The inhibitory effect of LL-37 on growth of CA at 180 min was as significant in preterm cord blood as it was in term cord blood (Figure 5C).\n\nViability of CA as measured in CFU is plotted on the y-axis relative to the incubation time in panels (A) and (B). (A) LL-37 but not MBL inhibits growth of CA in preterm blood at 1 min, 45 min and 180 min. (B) Significant LL-37 inhibition of SE growth in term blood at 1 min, 45 min and at 180 min. (C) Summary of LL-37 effects on SE growth in term and preterm cord blood at 180 min, demonstrating an equally pronounced inhibitory effect of LL-37 on CA growth in term and preterm cord blood. The inoculum (1x104 CFU/ml) at time-point “0” is plotted at 0.1 min. Term N = 20; preterm N = 8. Statistical analysis employed two-way ANOVA with Dunnett’s (A, B) or Sidak’s (C) post hoc test. ** p<0.0005. All graphs depict mean and error with SEM.\n\n\nDiscussion\n\nIn this study we have, to our knowledge for the first time, characterized the antimicrobial activity of exogenous LL-37 and rMBL when added to human preterm and term cord blood in vitro. While some studies suggest that relatively low serum MBL or LL-37 levels are associated with a risk of specific infections9,12,22–25, to our knowledge, including PubMed search as of date 2/24/18 using the term “LL-37” and “cord blood”, or “mannose binding lectin” and “cord blood”, none have measured the activity of these APPs when added to preterm or term newborn blood.\n\nOf note, preterm cord blood demonstrated a lower killing capacity against SA and SE than term cord blood. To our knowledge, this has not been demonstrated previously. As killing may be both extracellular and/or intracellular, this impairment in killing may reflect known deficits in plasma APP content with GA16 and/or impaired preterm neutrophil function, such as reduced chemotaxis and chemokinesis26.\n\nIn our cohort of newborns, MBL levels were markedly variable among both preterm and term cord blood samples and thus did not seem to correlate with GA (Figure 2A), consistent with studies demonstrating that cord blood MBL levels most closely reflect MBL genotype distribution rather than GA20.\n\nIn our study, MBL, at the concentrations tested in hirudinated whole blood, did not inhibit growth of SA, SE or CA in term or preterm cord blood. MBL in a sub-analysis of basal MBL levels, did exert modest fungistatic activity against CA in term newborn blood.\n\nLL-37 demonstrated significant antimicrobial and antifungal activity towards SE, SA and CA in term cord blood. It also demonstrated strong antimicrobial effects against SE and antifungal effects against CA in preterm cord blood. LL-37 generally exerted lesser antibacterial activity in preterm than in term blood, suggesting that it may act together with other host defense components that increase with GA. Of note, amongst other APPs, LL-37 levels are expressed in human breast milk, which demonstrated bacterial growth inhibitory effects towards both SA and SE, with activity towards SE increasing with the postnatal age of the breast milk expression27. LL-37 has previously been demonstrated to be a potent antimicrobial in adult peripheral blood28.\n\nOur study featured several strengths, including the use of a species- and GA-specific human whole blood assay system that is: (a) relatively physiological, (b) has been predictive of APP activity in vivo29, and (c) enables blood samples from the same individual to be assayed in both control and treatment conditions, including testing across a time range to characterize kinetic effects, thereby enhancing statistical power via paired analyses. Our study also has several limitations including: (a) relatively greater number of cord blood samples from term study participants (N = 22) than from preterm participants (N = 8), limiting the power to detect age-specific differences; (b) an absence of measurement of endogenous LL-37 levels due to sample and logistical limitations; and (c) limitations of the whole blood assay which, although it is often predictive, does not perfectly model in vivo conditions, including blood flow and endothelial interactions.\n\nIn conclusion, rMBL exhibited very modest fungistatic properties when added to term cord blood with high baseline MBL levels. By contrast, LL-37 inhibited the growth of SA, SE and CA in term cord blood, and SE and CA in preterm cord blood. To the extent that our in vitro system is relevant in vivo, LL-37 and its congeners30, such as immunoglobulin-based constructs that enhance half-life, may be promising agents to prevent and/or treat neonatal sepsis. Further translational studies of LL-37 designed to take into account both the pathogen identity and GA of the target population are warranted.\n\n\nData availability\n\nDataset 1. The complete raw data for the study, organized per figure. DOI: 10.5256/f1000research.14736.d20331721.", "appendix": "Competing interests\n\n\n\nThis work was funded by Shire Pharmaceuticals in the context of a sponsored research agreement.\n\n\nGrant information\n\nThis work was funded by Shire Pharmaceuticals in the context of a sponsored research agreement. OL’s laboratory is supported by U.S. National Institutes of Health (NIH) grants 1R01AI100135-01, and 3R01AI067353-05S1, the National Institutes of Allergy and Infectious Diseases (NIAID), NIH, Department of Health and Human Services, NIH UO1 award Molecular Mechanisms of Combination Adjuvants (1U01AI124284-01), Adjuvant Discovery Program Contract No. HHSN272201400052C, the NIH (NIAID) Human Immunology Project Consortium award U19AI118608 as well as Global Health (OPPGH5284) and Grand Challenges Explorations (OPP1035192) awards from the Bill & Melinda Gates Foundation and an internal BCH award to the Precision Vaccines Program. FB was supported by UniNA and Compagnia di San Paolo, in the frame of Programme STAR.\n\n\nAcknowledgments\n\nWe thank Jorge Velarde, MD, PhD (Infectious Diseases, Boston Children’s Hospital) for providing the LL-37 peptide, Julia Koehler, MD (Infectious Diseases, Boston Children’s Hospital) for providing the C. albicans strain and advice regarding culture, William Nauseef, MD from the University of Iowa Research Park for providing us with the S. aureus USA 300 strain and Kinga Smolen, PhD as well as other members of the Levy lab for helpful discussions regarding this manuscript.\n\n\nReferences\n\nWynn JL: Defining neonatal sepsis. Curr Opin Pediatr. 2016; 28(2): 135–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBone RC: The pathogenesis of sepsis. Ann Intern Med. 1991; 115(6): 457–69. PubMed Abstract \| Publisher Full Text\n\nDong Y, Speer CP: Late-onset neonatal sepsis: recent developments. Arch Dis Child Fetal Neonatal Ed. 2015; 100(3): F257–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoghossian NS, Geraci M, Edwards EM, et al.: Morbidity and Mortality in Small for Gestational Age Infants at 22 to 29 Weeks' Gestation. Pediatrics. 2018; 141(2): pii: e20172533. PubMed Abstract \| Publisher Full Text\n\nCollins A, Weitkamp JH, Wynn JL: Why are preterm newborns at increased risk of infection? Arch Dis Child Fetal Neonatal Ed. 2018; pii: fetalneonatal-2017-313595. PubMed Abstract \| Publisher Full Text\n\nBattersby AJ, Khara J, Wright VJ, et al.: Antimicrobial Proteins and Peptides in Early Life: Ontogeny and Translational Opportunities. Front Immunol. 2016; 7: 309. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPammi M, Suresh G: Enteral lactoferrin supplementation for prevention of sepsis and necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev. 2017; 6: Cd007137. PubMed Abstract \| Publisher Full Text\n\nVerjans ET, Zels S, Luyten W, et al.: Molecular mechanisms of LL-37-induced receptor activation: An overview. Peptides. 2016; 85: 16–26. PubMed Abstract \| Publisher Full Text\n\nHu Z, Murakami T, Suzuki K, et al.: Antimicrobial cathelicidin peptide LL-37 inhibits the pyroptosis of macrophages and improves the survival of polybacterial septic mice. Int Immunol. 2016; 28(5): 245–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHancock RE, Diamond G: The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol. 2000; 8(9): 402–10. PubMed Abstract \| Publisher Full Text\n\nAuriti C, Prencipe G, Moriondo M, et al.: Mannose-Binding Lectin: Biologic Characteristics and Role in the Susceptibility to Infections and Ischemia-Reperfusion Related Injury in Critically Ill Neonates. J Immunol Res. 2017; 2017: 7045630. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIsraëls J, Frakking FN, Kremer LC, et al.: Mannose-binding lectin and infection risk in newborns: a systematic review. Arch Dis Child Fetal Neonatal Ed. 2010; 95(6): F452–61. PubMed Abstract \| Publisher Full Text\n\nEick S, Puklo M, Adamowicz K, et al.: Lack of cathelicidin processing in Papillon-Lefèvre syndrome patients reveals essential role of LL-37 in periodontal homeostasis. Orphanet J Rare Dis. 2014; 9: 148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHartz A, Pagel J, Humberg A, et al.: The association of mannose-binding lectin 2 polymorphisms with outcome in very low birth weight infants. PLoS One. 2017; 12(5): e0178032. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKai-Larsen Y, Gudmundsson GH, Agerberth B: A review of the innate immune defence of the human foetus and newborn, with the emphasis on antimicrobial peptides. Acta Paediatr. 2014; 103(10): 1000–8. PubMed Abstract \| Publisher Full Text\n\nStrunk T, Doherty D, Richmond P, et al.: Reduced levels of antimicrobial proteins and peptides in human cord blood plasma. Arch Dis Child Fetal Neonatal Ed. 2009; 94(3): F230–1. PubMed Abstract \| Publisher Full Text\n\nFrakking FN, Brouwer N, Zweers D, et al.: High prevalence of mannose-binding lectin (MBL) deficiency in premature neonates. Clin Exp Immunol. 2006; 145(1): 5–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKronforst KD, Mancuso CJ, Pettengill M, et al.: A neonatal model of intravenous Staphylococcus epidermidis infection in mice <24 h old enables characterization of early innate immune responses. PLoS One. 2012; 7(9): e43897. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFonzi WA, Irwin MY: Isogenic strain construction and gene mapping in Candida albicans. Genetics. 1993; 134(3): 717–28. PubMed Abstract \| Free Full Text\n\nGrumach AS, Ceccon ME, Rutz R, et al.: Complement profile in neonates of different gestational ages. Scand J Immunol. 2014; 79(4): 276–81. PubMed Abstract \| Publisher Full Text\n\nScheid A, Li N, Jeffers C, et al.: Dataset 1 in: Antimicrobial peptide LL-37 and recombinant human mannose-binding lectin express distinct age- and pathogen-specific antimicrobial activity in human newborn cord blood in vitro. F1000Research. 2018. Data Source\n\nAuriti C, Prencipe G, Inglese R, et al.: Role of mannose-binding lectin in nosocomial sepsis in critically ill neonates. Hum Immunol. 2010; 71(11): 1084–8. PubMed Abstract \| Publisher Full Text\n\nde Benedetti F, Auriti C, D'Urbano LE, et al.: Low serum levels of mannose binding lectin are a risk factor for neonatal sepsis. Pediatr Res. 2007; 61(3): 325–8. PubMed Abstract \| Publisher Full Text\n\nSchlapbach LJ, Aebi C, Fisch U, et al.: Higher cord blood levels of mannose-binding lectin-associated serine protease-2 in infants with necrotising enterocolitis. Pediatr Res. 2008; 64(5): 562–6. PubMed Abstract \| Publisher Full Text\n\nKielgast S, Thiel S, Henriksen TB, et al.: Umbilical cord mannan-binding lectin and infections in early childhood. Scand J Immunol. 2003; 57(2): 167–72. PubMed Abstract \| Publisher Full Text\n\nBirle A, Nebe CT, Hill S, et al.: Neutrophil chemotaxis in cord blood of term and preterm neonates is reduced in preterm neonates and influenced by the mode of delivery and anaesthesia. PLoS One. 2015; 10(4): e0120341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrend S, Strunk T, Hibbert J, et al.: Antimicrobial protein and Peptide concentrations and activity in human breast milk consumed by preterm infants at risk of late-onset neonatal sepsis. PLos One. 2015; 10(2): e0117038. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlalwani SM, Sierigk J, Herr C, et al.: The antimicrobial peptide LL-37 modulates the inflammatory and host defense response of human neutrophils. Eur J Immunol. 2010; 40(4): 1118–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLevy O: Therapeutic potential of the bactericidal/permeability-increasing protein. Expert Opin Investig Drugs. 2002; 11(2): 159–67. PubMed Abstract \| Publisher Full Text\n\nWarren HS, Matyal R, Allaire JE, et al.: Protective efficacy of CAP18106-138-immunoglobulin G in sepsis. J Infect Dis. 2003; 188(9): 1382–93. PubMed Abstract \| Publisher Full Text" }	[ { "id": "35932", "date": "20 Jul 2018", "name": "Cinzia Auriti", "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 extremely interesting research paper, well conducted from the methodological point of view and the statistical processing of data, well written and with well documented results.\n\nThe idea of administering recombinant MBL to newborns with a MBL deficit is not new and could open many unconventional ways in treating neonatal infections. Therefore, studies that increase biological knowledge in this area are very relevant.\n\nThe observation to be made is that the side effects of MBL are not yet known exactly. MBL, on the one hand, promotes the defense against infections, but on the other if in excess, could by itself generate tissue damage, thanks to induction activity of complement system. This problem is more evident in newborns, who have little biological ability to contain inflammatory up regulation. So until today we do not know if we can safely administer recombinant MBL to human neonates without side effects.\n\nThat said, which could be cited in discussion, the study is experimental, of great interest because it adds important knowledge (type of bacteria, relationships with prematurity and baseline MBL levels) and in my opinion can be published.\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": "3846", "date": "20 Jul 2018", "name": "Annette Scheid", "role": "Author Response", "response": "We thank Dr. Auriti, a renown expert in neonatology as well as neonatal immunology and sepsis biology for her support in publishing our proposed article." } ] }, { "id": "36281", "date": "14 Sep 2018", "name": "Jerrold Weiss", "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 broader goals (seeking ways to enhance possibly limiting host defense capacities of pre-term and term newborns) are important, the experimental approach for these target samples (though not more generally) is novel, some (but not all) of the limits of this approach recognized, and the microbial targets and exogenously added host-derived (via recombinant form) reasonable. Of note, for experiments of this nature, the general reproducibility of the observations are striking. The description of the assay protocol should provide a more complete description of the \"physical format\" of the incubations (e.g., vessels used; type of sample shaking (or not)) that could impact, especially, the participation of the cellular elements of the blood samples that are likely important in the handling and outcome of these infections in vivo.\nThe main \"weakness\" of this study is that the findings seem not surprising and not leading to an obvious next chapter unless other test host agents come to mind and/or become available. The conclusions of the authors, I think, reflect a similar view. The demonstration of lower killing capacity of preterm vs term cord blood against the two staphylococcal species tested is a new finding that may be important but deserves a more thorough characterization. It is not surprising that this difference could also impact the potential efficacy of the added agents as shown in Figs. 3-5. How that works would be of interest and potential value. In the end, the choice of MBL and LL-37 -- considering their limited effects in very high test doses -- may not have been optimal.\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/7-616
https://f1000research.com/articles/7-423/v1	04 Apr 18	{ "type": "Opinion Article", "title": "Towards precision medicine: The application of omics technologies in asthma management", "authors": [ "Chiara Scelfo", "Carla Galeone", "Francesca Bertolini", "Marco Caminati", "Patrizia Ruggiero", "Nicola Facciolongo", "Francesco Menzella", "Carla Galeone", "Francesca Bertolini", "Marco Caminati", "Patrizia Ruggiero", "Nicola Facciolongo", "Francesco Menzella" ], "abstract": "Asthma is a chronic obstructive respiratory disease characterised by bronchial inflammation. Its biological and clinical features have been widely explored and a number of pharmacological treatments are currently available. Currently several aspects of asthma pathophysiological background remain unclear, and this is crucial for the traditional asthma phenotype approach as well as for new endotype definition. In this scenario, the identification of new molecular and clinical biomarkers may be helpful in order to better understand the disease, define specific diagnostic tools and highlight relevant novel targets for pharmacology treatments. Omics technologies offer an innovative research tool for addressing the above mentioned goals. However, there is still a lot to do both in the fields of basic research and in the clinical application of these new technologies. Recently, genome-wide association studies, microRNAs and proteomics are contributing to enrich the available data for the identification of new asthma biomarkers. A precise approach to the patient with asthma, particularly with severe uncontrolled asthma, requires new and specific therapeutic targets, but also proper tools able to drive the clinician in tailoring the treatment. On the other hand, treatment response predictors are needed, particularly in the field of biological drugs, whose sustainability implies a correct and precise patient selection. Translating acquired knowledge about omics in clinical practice may address the unmet needs described above, but large-scale studies are required in order to confirm omics relevance and effectiveness in daily practice. Thus in our opinion the application of omics is still lagging in the real-life setting.", "keywords": [ "Severe asthma", "Omics sciences", "Inflammation", "Precision medicine" ], "content": "Introduction\n\nSeveral aspects of asthma heterogeneity both from a clinical and pathophysiological perspective still remain unclear. A number of treatment options have been developed over time, from the widely used corticosteroids to personalized approaches, including recently introduced biological therapies. The new classification of severe asthma is based on endotypes, whose definition relies on the features of the underlying inflammation. The endotypes define traditional phenotypes by describing their pathophysiological mechanisms1,2. Exploring endotypes and phenotypes supports the identification of specific molecular targets, which can be addressed by precision treatments such as biologic drugs. The management of severe asthma is benefitting from personalized medicine approaches based on the characterization of an increasing number of endotypes, which represent the targets of specific therapies3. These are mainly represented by the Th2-high subtype and the Th2-low subtype, characterized respectively by the presence of eosinophilic or neutrophilic/paucigranulocytic airway inflammation3. Currently targeted therapies for a number of Th2 – low endotypes are still lacking4. For this and other reasons, the need for increasing the effectiveness of personalized therapies opens the field to the omics approach.\n\nNew asthma phenotyping has led to a growing interest in targeted therapies. The search for new pharmacological targets has caused interest in understanding the pathophysiological and molecular mechanisms underlying asthma. So far, the majority of the available drugs target the Th2-cytokine pathway5.\n\nOmics technology supports precision medicine in identifying the most effective treatment for different clinical phenotypes, in contrast with the “one size fits all” approach. Indeed, omics sciences contribute to the definition of new biomarkers, which can be useful as hallmarks of a specific asthma endotype or phenotype, and relevant as novel targets for pharmacology treatments. In the field of molecular biology, omics is a neologism that indicates high-throughput experimental technologies providing the tools for comprehensively monitoring the disease processes at a molecular level. The suffix “ome” comes from “chromosome” and currently includes several biological fields such as genomics, transcriptomics, proteomics, metabolomics and epigenomics. Genomics and transcriptomics have been used to identify genes associated with asthma severity (Figure 1). Recent genome-wide association studies (GWAS) have shed light on distinct pathways that contribute to asthma inflammation. Genes such as HLA, IL13, IL33, thymic stromal lymphopoietin (TSLP) involved in Th2 pathway, IL-1 receptor–like 1 (IL1RL1), encodes ST2, and the receptor for IL-33 are associated with asthma onset. In contrast, it is well-known that the risk of childhood asthma is associated with the 17q21 locus encoding the ORMDL3 and GSDML genes6,7. Transcriptomics are focused on microRNAs (MiRNAs). MiRNAs are small non-coding single strand RNA chains involved in post-transcriptional regulation processes. MiRNAs play a key role in regulating cell functions as well as in modulating the inflammatory pathways. They may influence the single endotype profile in the complex asthma phenotype picture. MiRNAs can be collected through peripheral blood sampling, or, more invasively, through bronchial biopsies and induced sputum8,9. Under-expression of miRNA 192 in blood was investigated in a small study, in which asthma patients underwent an allergen inhalation challenge10. The relevance of miRNA as a biomarker is increasingly investigated. Different levels of miR-1248 serum expression in asthmatic vs non-asthmatic patients have been documented. In asthmatic patients miR-1248 is also involved in the regulation of IL-5 pathway10. Among the omics, circulating miRNA deserves a specific interest. Circulating miRNAs might be a non-invasive biomarker useful in asthma diagnosis and characterisation, as demonstrated in a recent study. According to the authors, a specific subset of circulating miRNAs (miR-125b, miR-16, miR-299-5p, miR-126, miR-206, and miR-133b) was found in patients with allergic rhinitis and asthma11.\n\nEpigenomics represent another relevant issue. The “methylome” (the set of DNA methylation patterns) has been increasingly investigated through highly sophisticated sequence-based assays. Epigenetic mechanisms could lead to the identification of new asthma biomarkers. Recently, an epigenetic association between serum IgE concentration and methylation at different loci derived from DNA of leukocites has been described. Methylation at these CpG islands differed significantly in isolated eosinophils between subjects with and without asthma and high IgE levels12.\n\nModern and advanced technologies, such as mass spectrometry, allow the detection of several proteins involved in the inflammatory mechanisms of asthma. Among the proteomics signatures characterized so far, Galectin-3 deserves to be mentioned. It has been demonstrated that this protein is expressed in omalizumab responders only. Furthermore galectin–3 seems to be associated with a more evident respiratory function improvement in asthmatic patients treated with omalizumab13.\n\nThe increasing interest in metabolomics, is mainly due to its prospective clinical applications. According to recent findings it is possible to define the metabolic profile through different samples including exhaled breath, urine, plasma and serum14. Currently a branch of metabolomics called “breathomics” focuses on volatile organic compounds (VOCs) from the respiratory tract. VOCs represent potential non-invasive metabolic biomarkers, particularly in the diagnosis and monitoring of pulmonary diseases including asthma15. Moreover, an electronic nose able to discriminate asthmatic from healthy controls by detecting different VOCs in exhaled breath has been developed15,16. Therefore, metabolomics could play a key role in identifying biomarkers and improving asthma endotyping.\n\n\nConclusion\n\nThe application of omics technology in asthma is following other research fields, such as oncology17. Similarly, monoclonal antibodies (mAbs) for severe asthma have been recently introduced, while biological therapies addressing rheumatic diseases, solid tumors and blood cancer arrived more than a decade before. From 2006 to 2017 omalizumab was the only available treatment for severe allergic asthma. Only in recent years research and knowledge on new drugs has been developed to achieve new and more effective therapeutic options18. Though an increasing interest in omics technology, none of the omics signatures mentioned above have been translated into clinical practice. We believe that there is an urgent need for large-scale studies. Particularly, specific Randomized Controlled Trials would be necessary to definitively confirm the clinical relevance of omics and reinforcing omics’ role in searching for new biomarkers and prognostic factors. The need for correctly selecting the right mAb for the right patient is one of the key points in severe asthma management. The real challenge for researchers and clinicians in the “omics era” is therefore translating acquired knowledge into clinical practice in order to emphasize omics’ role in precision medicine and to predict response to treatments. Unfortunately, in our opinion we are still far from that scenario.\n\n\nData availability\n\nNo data is associated with this article.", "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\nWenzel SE: Asthma phenotypes: the evolution from clinical to molecular approcache. Nat Med. 2012; 716–725. PubMed Abstract \| Publisher Full Text\n\nStokes JR, Casale TB: Characterization of asthma endotypes: implications for therapy. Ann Allergy Asthma Immunol. 2016; 117(2): 121–25. PubMed Abstract \| Publisher Full Text\n\nSvenningsen S, Nair P: Asthma Endotypes and an Overview of Targeted Therapy for Asthma. Front Med (Lausanne). 2017; 4: 158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThomson NC: Novel approaches to the management of noneosinophilic asthma. Ther Adv Respir Dis. 2016; 10(3): 211–34. 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Am J Clin Exp Immunol. 2012; 1(2): 154–165. PubMed Abstract \| Free Full Text\n\nYamamoto M, Sing A, Ruan J, et al.: Decreased miR-192 expression in peripheral blood of asthmatic individuals undergoing allergen inhalation challenge. BMC Genomics. 2012; 13: 655. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPanganiban RP, Wang Y, Howrylak J, et al.: Circulating microRNAs as biomarkers in patients with allergic rhinitis and asthma. J Allergy Clin Immunol. 2016; 137(5): 1423–32. PubMed Abstract \| Publisher Full Text\n\nLiang L, Willis-Owen SAG, Laprise C, et al.: An epigenome-wide association study of total serum immunoglobulin E concentration. Nature. 2015; 520(7549): 670–674. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRiccio AM, Mauri P, De Ferrari L, et al.: Galectin-3: an early predictive biomarker of modulation of airway remodeling in patients with severe asthma treated with omalizumab for 36 months. Clin Transl Allergy. 2017; 7: 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKelly RS, Dahlin A, McGeachie MJ, et al.: Asthma Metabolomics and the Potential for Integrative Omics in Research and the Clinic. Chest. 2017; 151(2): 262–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Schee MP, Palmay R, Cowan JO, et al.: Predicting steroid responsiveness in patients with asthma using exhaled breath profiling. Clin Exp Allergy. 2013; 43(11): 1217–25. PubMed Abstract \| Publisher Full Text\n\nDragonieri S, Schot R, Mertens BJ, et al.: An electronic nose in the discrimination of patients with asthma and controls. J Allergy Clin Immunol. 2007; 120(4): 856–62. PubMed Abstract \| Publisher Full Text\n\nKumar M, Choudhury Y, Ghosh SK, et al.: Application and optimization of minimally invasive cell-free DNA techniques in oncogenomics. Tumour Biol. 2018; 40(2): 1010428318760342. PubMed Abstract \| Publisher Full Text\n\nHumbert M, Busse W, Hanania NA: Controversies and opportunities in severe asthma. Curr Opin Pulm Med. 2018; 24(1): 83–93. PubMed Abstract \| Publisher Full Text" }	[ { "id": "32719", "date": "24 Apr 2018", "name": "Claudio Micheletto", "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 heterogeneity and complexity of the asthma syndrome necessitates a different approach to its management. The use of clinical features and physiological and inflammatory data is no longer sufficient. Omics data and clustering will provide a greater chance of phenotyping asthma according to the mechanisms driving the disease in each phenotype, from which a composite set of biomarkers could be used to define and categorise the endotypes. This will help to develop personalised medicine for asthma that will allow for more precise treatment and also provide a source of novel targets and hence new treatments for each defined endotype. It is high time that personalised medicine be applied to the whole spectrum of asthma.\n\nThe application proposed by the authors is very promising, because it allows a precise phonotyping of asthmatic patients and a correct choice of therapeutic agents, in particular as regards the treatment with biologicals.\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": "3641", "date": "08 May 2018", "name": "Chiara Scelfo", "role": "Author Response", "response": "Dear Dr. Micheletto,We thank you for agreeing to review our manuscript. We also thank you for your valuable and positive comments. This is an incentive for us to try to improve our work.My best regards,Chiara Scelfo and coworkers" } ] }, { "id": "33170", "date": "10 May 2018", "name": "Mauro Maniscalco", "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\nBronchial asthma is a complex and heterogeneous pathology due to multiple mechanisms that are not present in all patients at any given time-point, or in the same patient at different time-points.\nFurthermore this disorder is not characterized by a single biomarker, but by a panel of biomarkers which describe its molecular aspects.\nIn the last years, “omics” sciences have improved disease phenotyping by linking the molecular mechanisms to the clinical field.\nIn their opinion article Scelfo and colleagues discussed about the application of omics sciences in the asthma characterization.\nThe topic is very interesting although I have some concerns regarding some aspects of the article:\nFirstly, the main concept of omics sciences, such as epigenomics and metabolomics, should be explained in a more extensive manner.\nFurthermore, several other proteins other than galectin- 3 are associated to severe asthma. I suggest to include other examples.\nOther matrixes (please prefer matrix to sample) have been used to metabolomics analysis in asthma such as exhaled breath condensate. This part should be extended, considered recent findings on the possibility to phenotype asthmatic patients using this method.\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/7-423
https://f1000research.com/articles/6-1978/v1	08 Nov 17	{ "type": "Research Article", "title": "Association of adverse childhood experiences with functional identity and impulsivity among adults: a cross-sectional study", "authors": [ "Muhammad Haaris Sheikh", "Sadiq Naveed", "Ahmed Waqas", "Ihtisham Tahir Jaura", "Muhammad Haaris Sheikh", "Sadiq Naveed", "Ihtisham Tahir Jaura" ], "abstract": "Introduction: The present study explores the association of adverse childhood experiences with impulsivity and functional identity among Pakistani adults. Methods: In this cross-sectional study, 260 Pakistani medical students aged 18 and above were approached. A consent form, a questionnaire on sociodemographic characteristics, and an English versions of the Adverse Childhood Experiences (ACE) scale, Functions of Identity scale (FIS) and Barratt’s Impulsiveness Scale (BIS-11) was employed in this study. All data were analyzed in SPSS v. 20. Results: A total of 122 (52.6%) of respondents had experienced at least one adverse childhood experience. According to linear regression analysis, ACE scores were significantly associated with increasing age, increasing order in birth, lower scores on functional identity structure and non-planning impulsivity, and higher scores on future (functional identity) and motor impulsivity. Conclusions: A high proportion of Pakistani medical students reported adverse childhood experiences, which lead to impulsive behaviors and poor functional identities.", "keywords": [ "adverse childhood", "impulsivity", "identity", "Pakistan", "abuse" ], "content": "Introduction\n\nThe Centers for Disease Control and Prevention (CDC) report a high prevalence of physical (28%) and sexual abuse (21%) associated with an unstable living environment among the American youth1. Previous studies demonstrate a significant relationship between experience of abuse and physical, behavioral and social problems among the youth1. Although there is abundant data exploring the prevalence of adverse childhood experiences in higher income countries, in low and middle income countries (LAMI) data is more scarce2 Moreover, a paucity of data has been identified in the LAMI, necessitating the need to transculturally translate the impact of adverse childhood events (ACEs) on social, cognitive and emotional impairment and adoption of high risk behaviors3.\n\nChildhood emotional mistreatment; particularly emotionally abusive acts, has been found to be associated with increased odds of lifetime diagnoses of several mental disorders4. The early, prolonged, and severe trauma can also increase impulsivity, diminishing the capacity of the brain to regulate emotions. Neurobiological studies show that childhood mistreatment leads to failure of inhibitory processes ruled by the frontal cortex over a fear-motivated hyper-responsive limbic system5. Therefore, impulsivity is a double edged sword, presenting itself as sequela of trauma as well as a risk factor for the development of a pathological response to trauma6. Many psychiatric disorders feature impulsivity, including substance-abuse disorders, attention deficit hyperactivity disorder, borderline personality disorder, conduct disorder and mood disorders. Impulsivity has also been associated with suicidal behaviors within various psychiatric populations exhibiting low serotonergic activity7. In mental health disorders especially substance use disorders, superimposition of the behavioral aftermaths of ACEs on impulsivity potentiate the risk of alcohol abuse by many folds8.\n\nSimilarly, previous studies have also established an association between ACEs and development of identity in adolescence. Development of a stable identity is a major developmental task, with its changing facets responsible for shaping the attachment styles and self-esteem in adolescence9,10. Serafini and Adams describe the importance of identity in providing structure for higher self-esteem and positive self-image; providing the goals necessary for self-direction11. This provides a sense of free will; harmony for social and academic adjustment; and future orientation that manifests as achievements in academia, aspirations and determination11. To address the gaps in scientific literature, the present study explores the association of adverse childhood experiences with subsequent impulsivity and functional identity among Pakistani adults.\n\n\nMethods\n\nThis study was designed as a cross-sectional study, where 260 medical students aged 18 and above and currently enrolled in King Edward Medical University and CMH Lahore Medical College & Institute of Dentistry, both in Lahore, were conveniently interviewed from April to May, 2017. Institutional review board approval was sought and obtained from the Ethical Review Board of CMH Lahore Medical College, Pakistan (approval number: 21/ERC/CMHLMC). A consent form, an anonymous questionnaire on sociodemographic characteristics, and English versions of the Adverse Childhood Experiences (ACE) scale, Functions of Identity scale (FIS) and Barratt’s Impulsiveness Scale (BIS-11) were employed in this study. Participation in this study was voluntary and written informed consent was obtained from all participants. The participants were ensured anonymity and that only group findings would be reported.\n\nThe Adverse Childhood Experiences (ACE) questionnaire is an important assessment tool that measures multiple types of abuse and adverse experiences that one may have encountered as a child1. Increasing scores correspond to a higher number of ACEs.\n\nThe Functions of Identity Scale (FIS) is a valid and reliable 5-point Likert scale, comprising 15 questions that assess five domains of psychological functions that identity serves for an individual: structure, goals, personal control, harmony and future11. Higher scores on these subscales correspond to a stronger sense of identity.\n\nBarratt’s Impulsiveness Scale (BIS-11) is a 30-item self-report Likert scale, with seven subscales; attention, motor, self-control, cognitive complexity, perseverance, and cognitive instability12. Higher scores on the scale or its subscales correspond to worsening impulsivity. All of these scales were found to be reliable in the present sample with following Cronbach’s α; ACE (0.71), FIS (0.86) and BIS-11 (0.78).\n\nAll data were analyzed in SPSS v. 21. Descriptive statistics were computed for the whole data. Frequencies were calculated and reported for ten domains of ACE, impulsivity and functions of identity. Multiple regression analysis was run to assess the association of impulsivity and functions of identity with ACEs, gender, age and socioeconomic status.\n\n\nResults\n\nA total of 232 medical students (232/260= 89.2%) responded to the surveys. The majority of them were females (n=188, 81%), with a mean age of 21.22 ± 1.31 years, mean number of siblings 3 ± 1.46, mean order of birth 1.94 ± 0.78 and a mean income greater than 30,000 PKR (n=208, 89.7%). Mean scores on subscales of Functional Identity Scale and Barratt’s Impulsiveness Scale are given in Table 1.\n\nA total of 122 (52.6%) respondents had experienced at least one ACE. Verbal, physical, sexual adverse events and poor support and affection from family were the most reported adverse events. Detailed statistics are presented in Table 2.\n\nAccording to multiple regression analyses (backward method), the predictors explained 14.2% of the variance in ACE scores (R2= 16.8%, adjusted R2= 14.2%, ANOVA F=6.435, P < 0.001). ACE scores were significantly associated with increasing age, increasing order in birth, lower scores on functional identity structure and non-planning impulsivity, and higher scores on future (functional identity) and motor impulsivity. Detailed statistics are presented in Table 3.\n\n\nDiscussion\n\nIn our study, adverse childhood experiences were significantly negatively associated with structure and positively with future subscales of the functional identity scale. Providing structure is a major function of one’s identity, deprivation of this results in poor self-esteem and negative self-image11. However, the negative association of adverse childhood experiences with the function of “future” was surprising. These adverse experiences may provide a better orientation in adulthood to fulfill one’s potential in academics and career in adulthood11.\n\nIndividuals reporting higher episodes of ACEs reported higher impulsivity, translating to a greater motor impulsiveness and a disrupted executive functioning among these individuals. However, these experiences were negatively associated with non-planning impulsiveness, representing an improved ability of forethought and planning for future12.\n\nThe results of this study should be generalized with caution. The cross-sectional nature of this study does not establish causality and temporality, therefore, future studies should employ a longitudinal study design.\n\n\nData availability\n\nDataset 1: Impulsivity and adverse childhood events. The dataset contains all variables pertaining to demographics, responses to Functional Identity Scale and Barrat’s Impulsiveness Scale. DOI, 10.5256/f1000research.13007.d18267013.\n\n\nConsent\n\nParticipation in this study was voluntary and written informed consent was obtained from all participants. The participants were ensured anonymity and that only group findings would be reported.", "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\nFelliti V, Anda R: The Adverse Childhood Experiences (ACE) study. Atlanta, GA Center of Disease Control and Prevention. 1997. Reference Source\n\nIram Rizvi SF, Najam N: Parental Psychological Abuse toward children and Mental Health Problems in adolescence. Pak J Med Sci. 2014; 30(2): 256–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSubstance Abuse and Mental Health Services Administration: Adverse Childhood Experiences. 2017, [cited 2017 Sep 3]. Reference Source\n\nTaillieu TL, Brownridge DA, Sareen J, et al.: Childhood emotional maltreatment and mental disorders: Results from a nationally representative adult sample from the United States. Child Abuse Negl. 2016; 59: 1–12. PubMed Abstract \| Publisher Full Text\n\nBraquehais MD, Oquendo MA, Baca-García E, et al.: Is impulsivity a link between childhood abuse and suicide? Compr Psychiatry. 2010; 51(2): 121–9. PubMed Abstract \| Publisher Full Text\n\nRoy A: Childhood trauma and impulsivity. Possible relevance to suicidal behavior. Arch Suicide Res. 2005; 9(2): 147–51. PubMed Abstract \| Publisher Full Text\n\nBrodsky BS, Malone KM, Ellis SP, et al.: Characteristics of borderline personality disorder associated with suicidal behavior. Am J Psychiatry. 1997; 154(12): 1715–9. PubMed Abstract \| Publisher Full Text\n\nDube SR, Anda RF, Felitti VJ, et al.: Adverse childhood experiences and personal alcohol abuse as an adult. Addict Behav. 2002; 27(5): 713–25. PubMed Abstract \| Publisher Full Text\n\nLuyckx K, Schwartz SJ, Berzonsky MD, et al.: Capturing ruminative exploration: Extending the four-dimensional model of identity formation in late adolescence. J Res Pers. 2008; 42(1): 58–82. Publisher Full Text\n\nSchwartz SJ, Beyers W, Luyckx K, et al.: Examining the light and dark sides of emerging Adults’ identity: A study of identity status differences in positive and negative psychosocial functioning. J Youth Adolesc. 2011; 40(7): 839–59. PubMed Abstract \| Publisher Full Text\n\nSerafini TE, Adams GR: Functions of identity: Scale construction and validation. Identity An Int J Theory Res. 2002; 2(4): 363–91. Publisher Full Text\n\nStanford MS, Mathias CW, Dougherty DM, et al.: Fifty years of the Barratt Impulsiveness Scale: An update and review. Pers Individ Dif. 2009; 47(5): 385–95. Publisher Full Text\n\nHaaris Sheikh M, Naveed S, Waqas A, et al.: Dataset 1 in: Association of adverse childhood experiences with functional identity and impulsivity among adults: a cross-sectional study. F1000Research. 2017. Data Source" }	[ { "id": "28427", "date": "12 Dec 2017", "name": "Usman Ali", "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 Pakistan, the researchers often have to deal with scarcity of existent data on virtually any subject. This article gives insight about adverse childhood experiences and its impact in terms of functional identity and impulsivity. However, there are a few points to be addressed.\n\nFirst, the title states, Association of adverse childhood experiences with functional identity and impulsivity among adults; a cross sectional study. However, the study population consists of medical students from early adulthood. This should be reflected in the title.\nSecondly, the religious and culturally constrained environment is different from other low and middle income countries. Hence, whenever a study in social sciences is conducted prior validity of used questionnaire should be established, which was not done in this study.\nFurthermore, in the conclusion the authors have stated a 'high' proportion of subjects who suffered from adverse childhood experiences. What were the control cut off values for high vs low proportion in this regard.\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?\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": "31204", "date": "19 Mar 2018", "name": "Syeda Fariha Iram Rizvi", "expertise": [ "Reviewer Expertise child abuse", "child and adolescent psychopathologies and problem behaviors", "family relationships" ], "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\nChildhood abuse is under recognized phenomena in Pakistan. This research is good effort to link childhood adverse experiences with impulsivity and identity. but there are few confusions/questions. Some detail of ACE questionnaire should be mentioned. Explain sub-variables in ACE in terms of questions. Is every single question of ACE is a separate variable? how we can say that if a person say yes to a single question that means he or she had a adverse childhood experience? Only Frequency and severity of an experience can determine its intensity.\nLiterature review is poor. Objectives and hypotheses are not mentioned in article.\nI have serious concerns regarding result/analysis section. Relationship should be analysed with correlational analysis first and then go for regression analysis to confirm the relation while identifying significant predictors.\nAnalysis is very much confused as I can’t understand that what variables are described as predictors and which one are outcome variables. According to Table no3 impulsivity and identity are describes as predictors/independent variables although according to title and literature ACE are predictors and impulsivity and identity related variables are outcome variables. So according to the purpose of research results are wrong. If author has something else in mind please explain it.\nIf author will mention objectives and hypotheses and then give analyses according to hypotheses then reader can understand what actually author want to explore.\nDiscussion is poorly written. please relate your results with existing literature\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?\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": [ { "c_id": "3644", "date": "18 May 2018", "name": "Ahmed Waqas", "role": "Author Response", "response": "Response to Reviewer: Dear Ms. Rizvi, We are grateful to you for providing such a valuable feedback to our short report exploring the association of adverse childhood experiences with functional identity and impulsivity among adults. We have updated our manuscript in line with your comments, and firmly believe that it has improved the quality of our manuscript. We hope for your favorable response in due time. Best wishes, Dr. Ahmed Waqas Corresponding author Comment 1: Explain sub-variables in ACE in terms of questions. Is every single question of ACE is a separate variable? Response 1: We are grateful to you for your feedback. We have expanded the methodology section with details on ACE questionnaire. It now reads as: “The Adverse Childhood Experiences (ACE) questionnaire is an important assessment tool that measures multiple types of abuse and adverse experiences that one may have encountered as a child 1 . It assesses adverse childhood experiences related to abuse (physical, psychological and sexual); neglect (emotional and physical) and household dysfunction (alcoholism or drug use at home, loss of biological parent, mental illness in home, violent treatment by mother and imprisoned household member). Responses to the ACE are recorded on a dichotomous scale (yes/no) and then scores are summed with higher scores corresponding to a higher number of ACEs. It has exhibited adequate reliability (Cronbach’s alpha 0.6 to 0.8) and validity in previous study1.” Comment 2: how we can say that if a person say yes to a single question that means he or she had a adverse childhood experience? Only Frequency and severity of an experience can determine its intensity. Response 2: The Adverse Childhood Experiences scale is one of the most widely used scales globally. It has demonstrated adequate factor validity as well as reliability in previous studies. Based on these merits, we had opted to use this scale in our setting. Furthermore, it also yielded an acceptable reliability (alpha= 0.71) in our study. Please, also see: Felliti V, Anda R: The Adverse Childhood Experiences (ACE) study. Atlanta, GA Center of Disease Control and Prevention.1997. Reference Source Ford DC, Merrick MT, Parks SE, Breiding MJ, Gilbert LK, Edwards VJ, Dhingra SS, Barile JP, Thompson WW. Examination of the factorial structure of adverse childhood experiences and recommendations for three subscale scores. Psychology of violence. 2014 Oct;4(4):432. Comment 3: Literature review is poor. Objectives and hypotheses are not mentioned in article. Response 3: We partly agree with your comment. But please, do understand that our manuscript is a short report, bound by a word limit of 1000 words excluding tables and references. Due to constraints of word count, we had provided a summary of the recent literature. And therefore, could not expand the introduction section, which has a word count exceeding 1200 at present. We have updated our introduction with objectives of the study. It reads as: “To address the gaps in scientific literature, the present study explores the association of adverse childhood experiences with demographics, subsequent impulsivity and functional identity among Pakistani adults.” Comment 4: I have serious concerns regarding result/analysis section. Relationship should be analysed with correlational analysis first and then go for regression analysis to confirm the relation while identifying significant predictors. Analysis is very much confused as I can’t understand that what variables are described as predictors and which one are outcome variables. According to Table no3 impulsivity and identity are describes as predictors/independent variables although according to title and literature ACE are predictors and impulsivity and identity related variables are outcome variables. So according to the purpose of research results are wrong. If author has something else in mind please explain it. If author will mention objectives and hypotheses and then give analyses according to hypotheses then reader can understand what actually author want to explore. Response 4: Dear Ms. Rizvi, thank you so much for your guidance. We have updated our results with new analyses providing mean scores of ACE scale (representing severity of adverse experiences) and frequency of individual types of ACEs. We have also replaced regression analysis with partial correlations adjusted for gender, age, year of study, number of siblings and order in birth. The results section now reads: Mean score (SD) on the ACE scale was 1.37 (1.75). A significant proportion of respondents cited verbal abuse (34.5%), physical (22.0%), sexual (15.5%), poor family support (19.0%), neglect (9.9%), separation/divorce of parents (4.7%), and witnessed domestic abuse (11.2%), substance abuse (3.9%), mentally or suicidal patient in the family (11.2%) and criminal background (4.7%). ACE scores yielded a significantly positive association with cognitive stability, perseverance and motor impulsivity on the Barrat’s impulsivity scale. Whereas, it yielded negative association with structure and harmony subscales of the functional identity as well as cognitive complexity subscale of the impulsivity scale. Moreover, no significant correlation was found with gender (P= 0.07), number of siblings (P= 0.95) and order in birth (P=0.08) and hoursehold income (P= 0.21). Age of participants was positively associated with ACE scores (r= 0.15, P= 0.02). Comment 5: Discussion is poorly written. please relate your results with existing literature Response 5: We agree with your comment. But please, do understand that our manuscript is a short report, bound by a word limit of 1000 words excluding tables and references. Therefore, we have replaced our discussion section with conclusion and limitations." } ] } ]	1	https://f1000research.com/articles/6-1978
https://f1000research.com/articles/7-612/v1	18 May 18	{ "type": "Research Article", "title": "Incidence and risk factors for complications after definitive skeletal fixation of lower extremity in multiple injury patients: a retrospective chart review", "authors": [ "Thananit Sangkomkamhang", "Wilaiphorn Thinkhamrop", "Bandit Thinkhamrop", "Wongsa Laohasiriwong", "Wilaiphorn Thinkhamrop", "Bandit Thinkhamrop", "Wongsa Laohasiriwong" ], "abstract": "Background: The management of multiple injuries is complex. Type and timing of treatment for lower extremity fractures is a controversial subject. Although many studies have demonstrated the safety and effectiveness of early treatment, others have suggested that early definitive stabilization may cause complications, especially with chest and head injuries. The aim of this study was to determine the complications and effects of timing of fixation, and investigate risk factors for complications in multiple injuries patients with lower extremity fractures. Methods: A Retrospective chart review from Khon Kaen Trauma Registry between 2008 and 2015 were collected. All major complications were identified and collected for example acute respiratory distress syndrome (ARDS), acute kidney injury (AKI) and sepsis. The time to definitive skeletal fixation from initial injury was identified and analyzed with multiple logistic regression. Results: 1224 multiple injuries patients with lower extremity fractures were identified. The mean age was 34±19.5 years, 74.4% were male and 25.6% female. The mean time from initial injury to definitive operation was 55.7±53.9 hours. Complications occurred with 178 patients (14.5%), the most common of which were pneumonia, ARDS and AKI. After adjusting for sex, severity of injury, we found that the operation within 24-48 hours complication was 6.67 times less common than in the early treatment group (less than 24 hours) (95% CI: 3.03 to 10.00, P-value< 0.001). Conclusions: About 15% of the multiple injuries patients with lower extremity fracture had major complications. The optimal time for definitive fixation in lower extremity fractures to reduce complications was within 24-48 hours. We found that if we operated too early (before 24 hours) or more than 48 hours after the injury it could increase the morbidity and mortality.", "keywords": [ "Trauma registry", "emergency medical services", "retrospective cohort study", "surgery" ], "content": "Abbreviations\n\nARDS Acute respiratory distress syndrome\n\nAKI Acute kidney injury\n\nMOF Multiple organ failure\n\nAIS Abbreviated Injury Score\n\nISS New Injury Severity Score\n\nGCS Glasgow Coma Scale score\n\nPE Pulmonary embolism\n\nDVT Deep venous thrombosis\n\nOR Odds ratio\n\n95% CI 95% confidence interval\n\n\nIntroduction\n\nManagement of lower extremity injury in patient with multiple injuries, especially femoral fractures, is a complex situation. The debate on the optimal time to fixation is commonly over two strategies, 1) early definitive fixation where definitive fixation is within 24 hours after the injury, and 2) damage control orthopedics (DCO). The benefits of early definitive fixation are lower pulmonary complications, shorter length of stay and less morbidity1–4. On the other hand, some of evidences suggested that early fixation of long bones have more complications in patient with head and chest injuries5–7 and can induce inflammatory processes, hypoperfusion and “second hit injury”8–10.\n\nDCO involves an initial skeletal stabilization for the patients with multiple injuries and an unstable condition, followed by delayed definitive fixation11–14. DCO has some advantages over early fixation in pulmonary function, pain relief14 and prevents the complication of early definitive fixation from initiating an inflammatory process that may be followed by acute respiratory distress syndrome (ARDS), acute kidney injury (AKI) and multiple organ failure (MOF)15,16. Many sources support the benefits of DCO17. A meta-analysis from Robinson however, showed a protective effect of early definitive fixation from pulmonary complications when compared with DCO ( RR 0.30,0.22–0.40 90% CI)17. Further complicating the situation, data has also been reported indicating no difference in morbidity and mortality between early and late definitive fixation of long bones18.\n\nSine there is inconclusive evidence for the optimal time to perform definitive fixation of long bones, we conducted this study to determine the complications and effects of timing of fixation and investigate risk factors for complications in multiple injury patients with lower extremity fractures.\n\n\nMethods\n\nA retrospective cohort study using data from the Khon Kaen Trauma Registry conducted between January 2008 and November 2015, was performed.\n\nA total of 1,224 multiple injury patients with lower extremity fractures treated between 2008 and 2015 were reviewed. Complications were identified from medical records that mentioned ‘major complication’ for example pneumonia, ARDS, AKI, sepsis and multiple organ failure (MOF). Timing to definitive skeletal fixation of lower extremity from initial injury were identified and analyzed with multiple logistic regression.\n\nThe Khon Kaen Trauma Registry collected pre-hospitalization and hospitalization information from medical records (including injury condition, comorbidities, fracture type, treatment type, operations, complications and outcomes)\n\nThe inclusion criteria for this study were 1) closed or open diaphyseal fracture of the femur and tibia; 2) multiple injuries in at least 2 body region; 3) underwent a definitive treatment of the long bone fracture with internal fixation. The exclusion criteria were 1) patient with late admission or transferred from other hospitals; 2) pathological fracture caused by neoplasm or malignancy; 3) open fracture grade IIIB or IIIC classified by Gustilo et al.19 or 4) inadequate information in the medical record (Figure 1).\n\nThe information of covariates and potential confounding in The Khon Kaen Trauma Registry were collected to identify the association between time of treatment and complications. The potential confounders included age, sex, comorbidity, type of fracture, type of treatment, Abbreviated Injury Score (AIS) for each of the six anatomical body regions (head/neck, face, chest, abdomen, extremity/pelvis, and skin)20, New Injury Severity Score (ISS)21 and Glasgow Coma Scale score (GCS) on admission22. In order to identify the association between time of definitive fixation of long bone and complications we divided patients with time of definitive fixation into three groups: 1) 24 hours or less, 2) between 24–48 hours, and 3) 48 hours or more from time of admission, based on cut-off points from previous studies23,24. Timing to definitive fixation was recorded in number of days and hours after admission.\n\nThe primary outcome analyzed was major complications, including pneumonia, pulmonary embolism (PE), ARDS, sepsis, deep venous thrombosis (DVT), AKI, MOF and mortality.\n\nFor diagnosis of ARDS, we defined it as an acute onset of bilateral pulmonary infiltrates on chest radiography and a PaO2:FiO2 200 mm Hg for four days and had no evidence of pneumonia and cardiogenic pulmonary edema25,26. Acute kidney injury was defined by renal insufficiency which required hemodialysis27. Multiple organ failure (MOF) was identified as failure of more than one organ system28.\n\nBaseline characteristics and selected variables were analyzed using descriptive statistical method, categorical variables were presented as number and percent, continuous data were reported as mean, standard deviation (SD), median, minimum value and maximum value (Min: Max). The effect of time to definitive long bone fixation, potential confounding and major complications were analyzed by logistic regression. All significant factors were evaluated with logistic multivariate regression analysis to eliminate the effect of confounding factors. Results are reported with odds ratios (OR) and 95% confidence intervals (CIs). STATA 10 (Stata Corp., College Station, TX, USA) statistical software was used for analysis.\n\nThe current study utilized data from the database of Khon Kaen Trauma Registry between 2008 and 2015 which was conducted in a single hospital in Thailand. Permission for this purpose was obtained from the hospital before receiving an approval from the Institutional Review Board (IRB) of Khon Kaen University with the reference number of HE602122. The data analysis was performed after the permission gained from the registry and approval was obtained from the IRB.\n\n\nResults\n\nThe average age was 34.0 ±19.5 years. 910 patients (77.4%) were male and 314 (25.6%) were female. More than 50% of the patients (n= 785) were treated within 48 hours after admission. The average time to definitive fixation was 55.7±53.9 hours. The median time to definitive fixation was 35.0 hours (1.5–293.1). After categorizing the time to definitive fixation into three groups, it was found the highest proportion was in the group of delayed definitive fixation of more than 48 hours (38.7%). The mean Glasgow coma scale score was 14.3 ± 2.2. The mean Injury Severity Score was 8.5 ± 7.7. The mean Abbreviated Injury Scale was 2.6 ± 0.6. Femur fractures were the most common fractures (57.6%) (Table 1).\n\nOverall, complication were found in 178 patients (13.4%). Most of the complications were pulmonary complications, specifically, pneumonia (6.7%) and ARDS (5.8%). Other complications were acute kidney injury (2.2%) and sepsis/MOF (0.9%). Patients with definitive fixation performed between 24–48 hours had fewer major complications than other groups as shown in Table 2. Pneumonia was found in 82 patients in total, 41 of which had definitive fixation more than 48 hours after injury, 37 in <48 hours group, and only 4 patients in the group were the fixation was performed between 24–48 hours after injury (6.7 per 100 people per year (PPY) (10.3–24.1;95%CI). A similar pattern was found in AKI and ARDS with an incidence of 2.2 per 100 PPY and 5.8 per 100 PPY respectively.\n\n*Acute respiratory distress syndrome (ARDS), acute kidney injury (AKI), multiple organ failure (MOF),\n\nhrs = hours\n\nThe data in Table 3 shows the association between major complications and time to definitive fixation together with other covariates. Time to definitive fixation between 24–48 hours had a statistical significant effect on decreasing the risk of major complications. (OR=0.15; 95% CI: 0.08–0.27; p-value = <0.001) compared with other groups. Factors that significantly increase the risk of major complication are being over 65 years of age (OR=2.36; 95% CI: 1.20–4.67; p-value = 0.010), Glasgow Coma Scale (GCS) of more than 8 (OR=0.07; 95% CI: 0.04–0.14; p-value = <0.001), an Injury Severity Score (ISS) more than 18 (OR=6.24; 95% CI: 4.10–9.50; p-value = <0.001). The Abbreviated Injury Scale (AIS), abdominal injury and lower extremities injury are also significantly associated with increased risk of major complications (Table 3).\n\nMultivariable analysis, with multiple logistic regression and adjusted for potential confounders; GCS, AIS BR and type of fracture, found that the risk factors that are associated with major complications were time to definitive fixation being less than 24 hours, being aged more than 65 years and an ISS of more than 18 (Table 4). Receiving definitive fixation between 24–48 hours following injury led a statistical significant decrease in risk of major complication (Adjusted OR=0.18; 95% CI: 0.10–0.33; p-value = <0.001) when compared with those who received that treatment lower than 24 hours. Those 65 years and older had a statistical significant increased risk of major complication (Adjusted OR=3.3; 95% CI: 1.6–6.5; p-value = <0.001). An Injury Severity Score (ISS) of more than 18 causes a statistical significant increase risk of major complication (Adjusted OR =5.90; 95%CI = 3.80-9.18; p – value = <0.001)\n\n\nDiscussion\n\nEarlier studies have demonstrated the advantage of early definitive treatment of long bone fractures, especially in femoral fractures, over delayed fixation5,6. The DCO developed as a treatment option for multiple injury patients with long bone fractures, combines the advantages of early fixation and decreases the physiologic and inflammatory process after the major orthopedic procedure15,29. Despite DCO being associated with a shorter operative time and less blood loss than definitive fixation (IMN or plating), the retrospective study of 97 severe multiple injured patients with ISS more than 25 showed no difference in complication of ARDS and MOF when compared with early definitive fixation14.\n\nWe found that delayed treatment of more than 48 hours after admission significantly increased the risk of complications compared with treatment within 24 hours or 24–48 hours. In some situations, the definitive treatment may be delayed more than two weeks after admission due to an unstable condition and is associate with further complications. These findings reflected the effect of the timing of definitive long bone fixation, especially in femoral shaft fractures in patients with multiple trauma, and care should be taken to avoid delay of treatment of more than the 48 hours from admission, similar to the study of Morshed et al23,30. In a large cohort study among multiple injury patients with an ISS of more than 15 with femoral shaft fractures where they studied the effect of timing of definitive fixation of femoral shaft fractures, they showed an increased length of stay for patients treated within 48 to 120 hours compared with other groups, especially in patients with chest trauma (AIS > 2). This study found that patients treated within 24 hours have lower length of stay23. In our study, we founding performing definitive fixation 24 to 48 hours after admission to the hospital, has improved outcomes and survival rates.\n\nIn the study of multiple injury patients with femoral shaft fractures (ISS >18) definitive fixation between 2 to 4 days after injury was associated with higher inflammatory conditions and an increased rate of multi-organ failure compared with fixation 5 to 8 days after the injury10. Other studies, however, have shown different results, with higher mortality when the operation is performed within the 2 to 5 day after injury; both of these resulted in high morbidity. Our study results found that performing fixation within 24–48 hours decreased the complications29.\n\nMany studies have supported this evidence that patients with multiple injuries namely head injuries31, chest injuries2 or abdominal injuries30 have higher morbidity and a higher risk of complications. For multivariate analysis with associated severe head injury, chest injury, abdominal injury and lower extremity injury, especially femoral fractures, after adjusting for confounding, show a decreased morbidity rate if fixation is performed within 24 to 48 hour.\n\nIn our study, for patients with an unstable conditions who received early definitive fixation within 24 hours had increased inflammation which has the effect of increasing mortality rate32,33.\n\nA study on early fixation of femoral shaft fractures (less than 24 hours) with a hypoperfusion state (serum lactate, > 2.5 mmol/L) demonstrated a similar number of postoperative complications to our study.(8) Multiple injury without appropriate resuscitation produces a hypoperfusion state and increases the inflammatory response leading to end-organ injury34. Damage control orthopaedics (DCO) involves temporary fixation of the fracture until resuscitation of the patient is adequate and patients are stable for definitive fixation15,35,36. For this study we found the benefits of delaying definitive long bone fixation for at least 24 hours until patients were stable and then performing the definitive fixation within 24–48 hours after admission to prevent second hit injury.\n\nThe retrospective chart review has limitation were selection bias and information bias. Variation in the time from the injury scene to hospital, lack of data for patients transferred from other hospital, the accurate time to operate?, and in some cases the data for DCO being incomplete contribute to information bias. This study has no investigation of blood chemistry, inflammatory mediators due to the fact it was a retrospective review. Complex femoral fractures, especially in proximal femoral fractures and distal femoral fractures, are not be clearly identified as a which may be lead to delayed fixation due to the fracture configuration and not by patient’s condition, which may affect the data. Furthermore there was a large number of patients who were transferred from other hospitals and therefore were not included in the study.\n\nDespite many limitations, our study has a large sample size of patients with multiple injuries and lower extremity fractures. The data has been collect from the Khon Kaen Trauma Registry, a referral and level I trauma center. The large cohort size supports the generalizability of our findings. The Khon Kaen Trauma Registry also has significant data on potential confounders, which was used for analytical multivariate methods.\n\n\nConclusion\n\nNearly 20% of multiple injuries patients with lower extremity fractures had major complications. We found that the timing of definitive fixation of lower extremity fracture (especially with femoral fracture) in multiple injury patients is associate with major complications.\n\nThe optimal time for definitive skeletal fixation in lower extremity fractures with the least complications based on our analysis appears to be within 24–48 hours. Performing operation too early (before 24 hours) or too late (after 48 hours) is associated with an increase in complication rate and should be considered in patient management for improved outcome.\n\nThe results support a delayed or “damage control orthopedics” management over definitive fixation of long bone fracture in multiple injuries patients. In patients who are unstable, adequate resuscitation at least 24 hours to 48 hours before undergo definitive fracture fixation is necessary, due to the systemic inflammatory response and to avoid “second hit” from a major surgical procedure. The trauma center hospitals should manage their resources to guarantee time to definitive fixation and proper management of multiple injuries patients.\n\n\nData availability\n\nDataset 1: Raw data obtained from the Khon Kaen Trauma Registry between 2008 and 2015. 10.5256/f1000research.14825.d20337237", "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 author would like to take this opportunity to gratefully thank Assoc. Dr.Bandit Thinkhamro, Assoc. Dr.Wongsa Laohasiriwong, Dr.Wilaiphorn Thinkhamrop, and Mr.Nathapob Chaichaya who is a statistician in the Data Management and Statistical Analysis Center (DAMASAC) for their valuable advice and kind support throughout this research. Additionally, this research could have been successful without Dr. Chanchai Janworachaikul, M.D. who is the director of Khon Kaen Hospital for allowing us access to the data of the registry. Dr.Wittaya Chartbunchachai, M.D., and Dr.Somkid Lertsinudom, M.D are also thanked for their full support in this research.\n\n\nReferences\n\nNahm NJ, Como JJ, Wilber JH, et al.: Early appropriate care: definitive stabilization of femoral fractures within 24 hours of injury is safe in most patients with multiple injuries. J Trauma. 2011; 71(1): 175–85. PubMed Abstract \| Publisher Full Text\n\nPape HC, Auf'm'Kolk M, Paffrath T, et al.: Primary intramedullary femur fixation in multiple trauma patients with associated lung contusion--a cause of posttraumatic ARDS? J Trauma. 1993; 34(4): 540–7; discussion 547–8. 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PubMed Abstract \| Publisher Full Text\n\nCrowl AC, Young JS, Kahler DM, et al.: Occult hypoperfusion is associated with increased morbidity in patients undergoing early femur fracture fixation. J Trauma. 2000; 48(2): 260–7. PubMed Abstract \| Publisher Full Text\n\nGiannoudis PV, Smith RM, Bellamy MC, et al.: Stimulation of the inflammatory system by reamed and unreamed nailing of femoral fractures. An analysis of the second hit. J Bone Joint Surg Br. 1999; 81(2): 356–61. PubMed Abstract \| Publisher Full Text\n\nPape HC, Schmidt RE, Rice J, et al.: Biochemical changes after trauma and skeletal surgery of the lower extremity: quantification of the operative burden. Crit Care Med. 2000; 28(10): 3441–8. PubMed Abstract \| Publisher Full Text\n\nScalea TM, Boswell SA, Scott JD, et al.: External fixation as a bridge to intramedullary nailing for patients with multiple injuries and with femur fractures: damage control orthopedics. J Trauma. 2000; 48(4): 613–21; discussion 621–3. PubMed Abstract \| Publisher Full Text\n\nNowotarski PJ, Turen CH, Brumback RJ, et al.: Conversion of external fixation to intramedullary nailing for fractures of the shaft of the femur in multiply injured patients. J Bone Joint Surg Am. 2000; 82(6): 781–8. PubMed Abstract \| Publisher Full Text\n\nTaeger G, Ruchholtz S, Waydhas C, et al.: Damage control orthopedics in patients with multiple injuries is effective, time saving, and safe. J Trauma. 2005; 59(2): 409–16; discussion 417. PubMed Abstract\n\nTuttle MS, Smith WR, Williams AE, et al.: Safety and efficacy of damage control external fixation versus early definitive stabilization for femoral shaft fractures in the multiple-injured patient. J Trauma. 2009; 67(3): 602–5. PubMed Abstract \| Publisher Full Text\n\nO'Toole RV, O'Brien M, Scalea TM, et al.: Resuscitation before stabilization of femoral fractures limits acute respiratory distress syndrome in patients with multiple traumatic injuries despite low use of damage control orthopedics. J Trauma. 2009; 67(5): 1013–21. PubMed Abstract \| Publisher Full Text\n\nBosse MJ, MacKenzie EJ, Riemer BL, et al.: Adult respiratory distress syndrome, pneumonia, and mortality following thoracic injury and a femoral fracture treated either with intramedullary nailing with reaming or with a plate. A comparative study. J Bone Joint Surg Am. 1997; 79(6): 799–809. PubMed Abstract\n\nRobinson CM: Current concepts of respiratory insufficiency syndromes after fracture. J Bone Joint Surg Br. 2001; 83(6): 781–91. PubMed Abstract \| Publisher Full Text\n\nRixen D, Grass G, Sauerland S, et al.: Evaluation of criteria for temporary external fixation in risk-adapted damage control orthopedic surgery of femur shaft fractures in multiple trauma patients: \"evidence-based medicine\" versus \"reality\" in the trauma registry of the German Trauma Society. J Trauma. 2005; 59(6): 1375–94; discussion 94–5. PubMed Abstract \| Publisher Full Text\n\nGustilo RB, Anderson JT: Prevention of infection in the treatment of one thousand and twenty-five open fractures of long bones: retrospective and prospective analyses. J Bone Joint Surg Am. 1976; 58(4): 453–8. PubMed Abstract \| Publisher Full Text\n\nMacKenzie EJ, Steinwachs DM, Shankar B: Classifying trauma severity based on hospital discharge diagnoses. Validation of an ICD-9CM to AIS-85 conversion table. Med Care. 1989; 27(4): 412–22. PubMed Abstract \| Publisher Full Text\n\nOsler T, Baker SP, Long W: A modification of the injury severity score that both improves accuracy and simplifies scoring. J Trauma. 1997; 43(6): 922–5; discussion 925–6. PubMed Abstract \| Publisher Full Text\n\nGill M, Windemuth R, Steele R, et al.: A comparison of the Glasgow Coma Scale score to simplified alternative scores for the prediction of traumatic brain injury outcomes. Ann Emerg Med. 2005; 45(1): 37–42. PubMed Abstract \| Publisher Full Text\n\nMorshed S, Mikhail C, Miclau Iii T: Timing of Femoral Shaft Fracture Fixation Affects Length of Hospital Stay in Patients with Multiple Injuries. Open Orthop J. 2015; 9: 324–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrundage SI, McGhan R, Jurkovich GJ, et al.: Timing of femur fracture fixation: effect on outcome in patients with thoracic and head injuries. J Trauma. 2002; 52(2): 299–307. PubMed Abstract \| Publisher Full Text\n\nDushianthan A, Grocott MP, Postle AD, et al.: Acute respiratory distress syndrome and acute lung injury. Postgrad Med J. 2011; 87(1031): 612–22. PubMed Abstract \| Publisher Full Text\n\nKonstantinides SV, Torbicki A, Agnelli G, et al.: 2014 ESC guidelines on the diagnosis and management of acute pulmonary embolism. Eur Heart J. 2014; 35(43): 3033–69, 3069a–3069k. PubMed Abstract \| Publisher Full Text\n\nLopes JA, Fernandes P, Jorge S, et al.: Acute kidney injury in intensive care unit patients: a comparison between the RIFLE and the Acute Kidney Injury Network classifications. Crit Care. 2008; 12(4): R110. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDurham RM, Moran JJ, Mazuski JE, et al.: Multiple organ failure in trauma patients. J Trauma. 2003; 55(4): 608–16. PubMed Abstract \| Publisher Full Text\n\nPape HC, Grimme K, Van Griensven M, et al.: Impact of intramedullary instrumentation versus damage control for femoral fractures on immunoinflammatory parameters: prospective randomized analysis by the EPOFF Study Group. J Trauma. 2003; 55(1): 7–13. PubMed Abstract \| Publisher Full Text\n\nMorshed S, Miclau T 3rd, Bembom O, et al.: Delayed internal fixation of femoral shaft fracture reduces mortality among patients with multisystem trauma. J Bone Joint Surg Am. 2009; 91(1): 3–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu XY, Jiang M, Yi CL, et al.: Early intramedullary nailing for femoral fractures in patients with severe thoracic trauma: A systemic review and meta-analysis. Chin J Traumatol. 2016; 19(3): 160–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHofmann S, Huemer G, Kratochwill C, et al.: [Pathophysiology of fat embolisms in orthopedics and traumatology]. Orthopade. 1995; 24(2): 84–93. PubMed Abstract\n\nNast-Kolb D, Waydhas C, Gippner-Steppert C, et al.: Indicators of the posttraumatic inflammatory response correlate with organ failure in patients with multiple injuries. J Trauma. 1997; 42(3): 446–54; discussion 454–5. PubMed Abstract \| Publisher Full Text\n\nHarwood PJ, Giannoudis PV, van Griensven M, et al.: Alterations in the systemic inflammatory response after early total care and damage control procedures for femoral shaft fracture in severely injured patients. J Trauma. 2005; 58(3): 446–52; discussion 452–4. PubMed Abstract \| Publisher Full Text\n\nPape HC, Hildebrand F, Pertschy S, et al.: Changes in the management of femoral shaft fractures in polytrauma patients: from early total care to damage control orthopedic surgery. J Trauma. 2002; 53(3): 452–61; discussion 461–2. PubMed Abstract\n\nBernhard M, Becker TK, Nowe T, et al.: Introduction of a treatment algorithm can improve the early management of emergency patients in the resuscitation room. Resuscitation. 2007; 73(3): 362–73. PubMed Abstract \| Publisher Full Text\n\nSangkomkamhang T, Thinkhamrop W, Thinkhamrop B, et al.: Dataset 1 in: Incidence and risk factors for complications after definitive skeletal fixation of lower extremity in multiple injury patients: a retrospective chart review. F1000Research. 2018. Data Source" }	[ { "id": "34168", "date": "29 May 2018", "name": "Saradej Khuangsirikul", "expertise": [ "Reviewer Expertise Hip and knee surgery", "lower extremities' orthopaedic trauma" ], "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\nAs the authors mentioned, selection bias was an issue. Type, classification, and fracture geometry, those were not recorded, also the important factors associated with postoperative complications. Different experience of multiple surgeons should be mentioned for the information bias. The reasons of delayed definitive operations should be classified, such as, patient condition, surgeon intension, randomisation, or hospital facilities etc. Exactly, \"time from the injury scene to definitive fixation\" is more concerned duration and more appropriate than \"time from admission\".\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "34165", "date": "31 May 2018", "name": "Piya Kiatisevi", "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 reviewed large number of multiple injuries patients with lower extremity fracture and, in the abstract, concluded that the optimal time for definitive fixation to reduce complications was within 24-48 hours.\nHowever, the authors did not clarify the reason for the delay surgery which could be unstable patient condition or operation room availability or other reasons. Complications in these groups could be different and affected the patient outcome. Do we really need to speed up the improvement process of the patient condition to get fracture fixed in 24-48 hours? Can we conclude from this paper that definite fracture treatment before 24 hours is not recommended? For patients whose conditions are stable before 24 hours, should we wait until we get into 24-48 hours period to reduce the inflammatory process? For the conclusion “optimal time for definitive fixation to reduce complications was within 24-48 hours”, are the severity of the injuries, type of injuries, pre-operative conditions of patients, fracture configuration and operative time in this group similar when compare to those of <24 hours and > 48 hours groups? Should the authors do subgroup analysis on this point? The percentage of multiple injuries patients with lower extremity fracture who had major complications in the abstract is not equal those on the main manuscript, please correct.\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?\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/7-612
https://f1000research.com/articles/7-611/v1	18 May 18	{ "type": "Research Article", "title": "Prevalence of neurogenic pulmonary edema among patients who died from head injury – a retrospective chart review", "authors": [ "Erasmus E. Okello", "Janat Tumukunde", "Patience Atumanya", "Sam Kalungi", "Alex Muhindo", "Arthur Kwizera", "Erasmus E. Okello", "Janat Tumukunde", "Sam Kalungi", "Alex Muhindo", "Arthur Kwizera" ], "abstract": "Background: Neurogenic pulmonary edema is a less recognized complication of head injuries and is seldom considered in management protocols in most centers. We therefore conducted a study to determine the prevalence of Neurogenic Pulmonary Edema among patients who died from head injury at Mulago National Referral Hospital, Uganda. Methods: An observational study conducted from the 1st June to 31st August 2013, with ethical approval. We consecutively enrolled patients who died of head injuries in the hospital. Demographic data, duration of illness, severity of illness, and patient management instituted were gathered from patient medical files. Autopsy results of the brain, lungs, heart, liver and kidneys performed within 24 hours after death were reviewed. Results: Twenty-six patients who died from head injury were enrolled in this study over the study period. Only one patient had preexisting hypertension and obesity while the rest had no known premorbid medical history. Intracranial abnormalities including raised intracranial pressure had been detected by CT scan in 18/26 of the patients while others had a normal scan (1/26) or did not have a scan done (7/26). Features of pulmonary edema were detected in 76.9% (20/26) of the bodies on gross lung examination. Pulmonary edema was bilateral in 85% of the bodies. Congestion was also noted in the liver, heart and kidneys. Conclusions: Pulmonary edema is highly prevalent in head injury patients and needs to be critically recognized early in the formulation of a management plan, as it contributes to morbidity and secondary brain injury through respiratory embarrassment.", "keywords": [ "head injury", "neurogenic pulmonary edema" ], "content": "Introduction\n\nNeurogenic pulmonary edema (NPE) is an age-old clinical syndrome characterized by the acute onset of pulmonary edema following a significant central nervous system (CNS) insult1. NPE can occur after virtually any form of injury of the CNS, and is a potential early contributor to pulmonary dysfunction in patients with head injuries2. A myriad of CNS events, including spinal cord injury, subarachnoid hemorrhage, traumatic brain injury (TBI), intracranial hemorrhage, status epilepticus, meningitis, and subdural hemorrhage, have been associated with this syndrome3–6.\n\nThe exact mechanism is unknown as it is believed to be multi-pronged and data is sparse. The connection between CNS injury and hemodynamic dysfunction was first described by Harvey Williams Cushing in 19031. This was followed by reports of cases in which acute pulmonary edema developed after CNS insult resultant from status epilepticus1,7 and isolated head bullet wounds3. The etiology is thought to be a surge of catecholamines following the head injury that results in cardiopulmonary dysfunction8,9. Several cardiovascular events are likely to result from this stimulation resulting in capillary leak and eventual pulmonary edema1. Bahloul et al. identified features of left ventricular myocardial dysfunction in an echocardiographic study performed on previously healthy traumatic brain injury patients presenting with neurogenic pulmonary edema5.\n\nTBI is one of the world’s major causes of mortality and morbidity, particularly in the developing world, where motor vehicle and cyclist crashes are largely due to human error and poor roads10–13. With increasing industrialization of the developing world, there is a proportional increase in motorized trauma. Uganda is currently ranked second in the world in road traffic accidents after Ethiopia14. These in a country with a limited health budget, mean resources are stretched to the limit by the burden of care required by trauma patients. TBI forms the majority of trauma cases occurring, either in isolation or as part of multiple trauma14,15.\n\nThe neurosurgery Unit of Mulago NRH admits at least ten critically ill patients per day, while the intensive care unit (ICU) can only admit a maximum of four patients. The national critical care bed capacity is significantly limited for the enormous population in need16. An in-hospital mortality of 25% following severe TBI was reported in a retrospective study in Mulago NRH13 while Kwizera et al. found an ICU mortality rate of 45.3% from head injury nationally. Mortality was commonly due to hemorrhagic shock, respiratory failure and sepsis1. The majority of survivors are left with gross disabilities while out-of-hospital mortality post-discharge remains largely unknown16.\n\nAwareness of NPE may lead to early intervention in brain-injured patients. However, although NPE was identified over 100 years ago, it is still underappreciated in the clinical arena. Its sporadic and relatively unpredictable nature, and a lack of etiologic-specific diagnostic markers and treatment modalities, may in part be responsible for its poor recognition at the bedside1. Worse still, there is very little data on the subject. Current data estimate the prevalence of NPE following severe brain insults, like trauma, stroke, status epilepticus etc., to be about 50%17, rising up to 92% in fatal subarachnoid hemorrhage18, being directly related to duration since insult4. Most data were, however, based on reviews of hospital records and case studies.\n\nIn this study, we determined the prevalence of neurogenic pulmonary edema through postmortem studies of head injured patients and sought to establish its relationship to the different patterns of organ dysfunction in head injured patients. This study also indirectly assessed quality of care of neurosurgery patients in the hospital. There are no published data on a similar study to the best of our knowledge.\n\n\nMethods\n\nThis was an observational study carried out in Mulago National Referral and Teaching Hospital, Uganda, involving patients who died from head injury. We consecutively enrolled subjects into the study upon death over two months (1st June to 31st August 2013) Medical records were reviewed and data on patient demographics and progressive medical/surgical management collected. Particularly, data about diagnosis and diagnostic procedures, medication, fluid administration, ICU admission, mechanical ventilation, and surgical procedures carried out.\n\nConsent to enroll patients was gathered from relatives upon death between June and August, 2013. We recruited all patients who died from TBI and excluded patients with documented cardiac dysfunction, chronic renal or hepatic dysfunction. A medical record review was performed to collect pertinent information on demographic, diagnosis (clinical, radiological, laboratory), duration of injury (from time of injury to time of death), patient management (length of hospital stay, nursing care, fluid management, medications, surgical interventions, ICU admission, mechanical ventilation) up to time of death. Medications sought for included steroids, antibiotics, diuretics, vasopressors, and anticonvulsants.\n\nPostmortem studies were performed by a pathologist (KS) within 24 hours of death. Full physical examination of the bodies was done, and notable abnormalities documented. Features of edema were sought during the examination of the heart, lungs, brain and kidneys. Edema was defined by tissue congestion with interstitial fluid on gross examination. A calibrated beam balance was used to weigh the brain, lungs, kidneys and heart. Organ weights were compared to standardized normal ranges based on the European guidelines19\n\nBasic descriptive statistics were used to analyze demographic data and other study variables. Data were analysed in STATA 10.0 Logistic regression analysis was used to determine the association between different variables with presence of NPE. P-values <0.05 were considered statistically significant (Table 3). Data are presented as modes and means unless otherwise indicated.\n\n\nResults\n\nDuring the study period, twenty-six patients were enrolled in this study. No patient had a premorbid diagnosis of renal, liver or lung disease. Only one patient had preexisting hypertension and obesity but with no documented cardiac disease. No drug or transfusion reactions were noted during the patients’ treatment. Only one patient (3.9%) had aspiration pneumonia. Intracranial abnormalities including raised intracranial pressure had been detected by brain Computed Tomography (CT) scan in 18/26 of the patients. One patient’s brain CT scan showed no acute intracranial pathology while 26.9% (7/26) of patients did not have a brain CT scan performed (Table 1). Autopsy showed features consistent with pulmonary edema in 76.9% of patients, being bilateral in 85% of these. All the 15% in whom edema was unilateral had involvement of the right lung (Table 2).\n\nCT: Computed tomography, ICU: Intensive care unit, AKI: Acute kidney injury\n\nStandard normal organ weights at autopsy19:\n\n• Brain Weight: Male: 1365–1450g; Female: 1250–1275g\n\n• Lung Weight: Left: 420–600g; Right: 480–680g\n\n• Heart Weight: Male: 270–280g; Female: 250–280g\n\n• Kidneys: Left: 150g; Right: 190g\n\nCT: Computed Tomography; CHI: Closed head injury; OHI: Open head injury\n\nBrain CT findings included intracranial edema and hemorrhage in 69.2%. 14 (53.9%) of patients had concomitant brain contusions while only one did not reveal any radiological features of intracranial pathology. Only three (11.5%) patients had had chest x-rays performed which revealed features consistent with bilateral airspace disease (Table 1). All patients admitted to ICU received invasive artificial ventilation. Average daily total fluid input was 2 liters with an average overall measured fluid balance (total IV and oral fluid input minus outputs from drains (urine, GI) (Table 1) of +560 mL at time of death. No transfusion reactions were recorded. None of the patients required instrumentation of the chest wall. Five patients (30.8%) had received orthopedic surgical care, two (7.7%) had received surgical toilet for soft tissue injuries while an additional two patients had undergone exploratory laparotomy for blunt abdominal trauma (ruptured spleen and ruptured liver, respectively) (Table 1).\n\nFeatures of pulmonary edema were noted in 76.9% of patients. 85% of these showed evidence of pulmonary edema bilaterally. Additional injuries noted at autopsy included liver and splenic contusion in one patient each, musculoskeletal injuries (degloving injuries (7.7%), long bone fractures (30.8%). There was no evidence of spinal trauma at autopsy (Table 2). Organs from one patient aged 9 years were not weighted against the European standard to which organ weights were referenced.\n\n\nDiscussion\n\nThe true incidence of NPE after acute head injury is difficult to estimate because much of the information comes from small autopsy series or isolated case reports. In this postmortem study, we found prevalence of neurogenic pulmonary edema of 76.9%. In an autopsy and in-patient database study, Rogers et al. found a prevalence of neurogenic pulmonary edema of 32% in patients dying within the first 24 hours and up to 50% in 96 hours2.\n\nPulmonary dysfunction after acute brain injury is a common but poorly understood phenomenon. In classic medical literature, the causes of pulmonary dysfunction in patients with head injury include pneumonia, aspiration, and pulmonary embolus, while NPE is seldom recognized. NPE is a form of pulmonary edema that develops rapidly after a cerebral injury20. It has been described in trauma patients as parenchymal edema, hemorrhage, and congestion without evidence of chest trauma in patients with isolated head injury18,21.\n\nIn Uganda, over 20,000 road traffic crashes occur annually claiming many lives through multiple trauma, head injury being one of the leading causes of death14,15,22,23. However, the incidence of NPE had not previously been established. In the study reported here, we included only patients with traumatic head injury who died after admission and treatment in the hospital. In addition, because of a shortage of resources, it was not possible to carry out several specialized tests on the patients before death.\n\nPulmonary edema was bilateral in most patients studied and was predominantly right sided in those with unilateral edema. This relates closely with the theory of a generalized pulmonary vascular disorder in head injured patients which is related to the severity of insult24. The study by Rogers et al., however, describes a predominantly right sided edema presentation2. This difference could be related to the difference in severity of illness and quality of initial management instituted. Furthermore, our study applied an European standard to organ weight measurement which was validated in a different population and only gross lung examination was performed for evidence of pulmonary edema which could have contributed to disparity in measurements and findings.\n\nStudies on the other organs showed no significant weight increases. In addition, no significant structural abnormalities indicative of preexisting chronic conditions could be found on anatomical examination of these organs. This correlates well with the definition of neurogenic pulmonary edema in which there is no other significant organ injury in a patient with severe brain injury1,24,25. Edema was, however, seen in some dissections of the heart and kidneys that could be a result of multi-organ dysfunction as a result of critical illness. In addition, Bahloul et al. described myocardial dysfunction in neurogenic injury which could in part explain the pathophysiology of NPE5\n\nSevere TBI frequently presents with significant depression in level of consciousness compromising adaptive protective reflexes. Standard management for severe TBI patients includes ICU admission for intensive monitoring, interventions and supportive care such as invasive mechanical ventilation, among other therapies. Moreover permissive hypercapnia and tight control of blood carbondioxide tension forms an integral part of neurocritical care26,27. In our study however, only 26.9% of patients were managed in ICU. Consequently, only 26.9% of patients in this study had received mechanical ventilatory support despite 76.9% having potentially developed respiratory dysfunction due to pulmonary edema among other causes. This is partly explained by the shortage of ICU capacity nationally16 and underscores the pressing need for critical care services in the care of TBI patients.\n\nBrain CT scan is a minimum diagnostic tool. In our study, 26.9% of patients never had a brain CT scan performed. This in addition to limited surgical capacity leads to delay and missed opportunities. As a result, the mortality of head injury patients is very high, resulting perhaps in complications such as neurogenic pulmonary edema. Absence of complete antemortem investigation of the patients before death for pulmonary edema plus associated conditions as well as the small sample size makes it difficult to deduce statistically significant causal relationships in our study. However, strong inferences can be derived from association between severity of brain injury (as shown by need for ICU admission, type of injury) with NPE.\n\n\nConclusion\n\nWe conclude that NPE occurs frequently in head injury patients. The process of edema formation begins early in the clinical course and is isolated to the lung. NPE thus needs to be critically recognized early in the formulation of a management plan. The contribution of NPE to mortality and morbidity in these patients cannot be deduced from this study, as a causal association between pulmonary edema and mortality was not studied.\n\nApproval of the study protocol was obtained from Mulago National Referral and Teaching Hospital Ethics Board. Written informed consent to participate and publish patient records was obtained from relatives. Denial of consent was criteria for omission from the study. All authors vouch for the originality, completeness, accuracy of the data presented and for the fidelity of this report to the study protocol as detailed herein and copied to the Mulago National Referral and Teaching Hospital Ethics Board.\n\n\nData availability\n\nDataset 1: Extracted medical data from patients with neurogenic pulmonary edema following head injury 10.5256/f1000research.13750.d20280628", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no funding was involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors of this study wish to convey their deep appreciation to the Intensive care, neurosurgical and pathology units of Mulago National Referral and Teaching Hospital for support and permission to access medical records and perform this research. Additionally, we would like to thank Mrs. Catherine Kwizera for the statistical work done in summarizing and analyzing data of this study.\n\n\nReferences\n\nDavison DL, Chawla LS, Selassie L, et al.: Neurogenic pulmonary edema: Successful treatment with IV phentolamine. Chest. 2012; 141(3): 793–795. PubMed Abstract \| Publisher Full Text\n\nRogers FB, Shackford SR, Trevisani GT, et al.: Neurogenic pulmonary edema in fatal and nonfatal head injuries. J Trauma. 1995; 39(5): 860–6; discussion 866-8. PubMed Abstract \| Publisher Full Text\n\nFontes RB, Aguiar PH, Zanetti MV, et al.: Acute neurogenic pulmonary edema: Case reports and literature review. J Neurosurg Anesthesiol. 2003; 15(2): 144–150. PubMed Abstract \| Publisher Full Text\n\nKaufman HH, Timberlake G, Voelker J, et al.: Medical complications of head injury. Med Clin North Am. 1993; 77(1): 43–60. PubMed Abstract \| Publisher Full Text\n\nBahloul M, Chaari AN, Kallel H, et al.: Neurogenic pulmonary edema due to traumatic brain injury: evidence of cardiac dysfunction. Am J Crit Care. 2006; 15(5): 462–70. PubMed Abstract\n\nMartin AM Jr, Simmons RL, Heisterkamp CA 3rd: Respiratory insufficiency in combat casualties. I. Pathologic changes in the lungs of patients dying of wounds. Ann Surg. 1969; 170(1): 30–38. PubMed Abstract \| Free Full Text\n\nColice GL: Neurogenic pulmonary edema. Clin Chest Med. 1985; 6(3): 473–489. PubMed Abstract\n\nGrunsfeld A, Fletcher JJ, Nathan BR: Cardiopulmonary complications of brain injury. Curr Neurol Neurosci Rep. 2005; 5(6): 488–493. PubMed Abstract \| Publisher Full Text\n\nZhao H, Lin G, Shi M, et al.: The mechanism of neurogenic pulmonary edema in epilepsy. J Physiol Sci. 2014; 64(1): 65–72. PubMed Abstract \| Publisher Full Text\n\nHelmy A, Vizcaychipi M, Gupta AK: Traumatic brain injury: Intensive care management. Br J Anaesth. 2007; 99(1): 32–42. PubMed Abstract \| Publisher Full Text\n\nHeegaard W, Biros M: Traumatic Brain Injury. Emerg Med Clin North Am. 2007; 25(3): 655–678, viii. PubMed Abstract \| Publisher Full Text\n\nHaddad SH, Arabi YM: Critical care management of severe traumatic brain injury in adults. Scand J Trauma Resusc Emerg Med. 2012; 20: 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTran TM, Fuller AT, Kiryabwire J, et al.: Distribution and characteristics of severe traumatic brain injury at mulago national referral hospital in Uganda. World Neurosurg. 2015; 83(3): 269–277. PubMed Abstract \| Publisher Full Text\n\nHsia RY, Ozgediz D, Mutto M, et al.: Epidemiology of injuries presenting to the national hospital in Kampala, Uganda: Implications for research and policy. Int J Emerg Med. 2010; 3(3): 165–172. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKobusingye O, Guwatudde D, Lett R: Injury patterns in rural and urban Uganda. Inj Prev. 2001; 7(1): 46–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKwizera A, Dünser M, Nakibuuka J: National intensive care unit bed capacity and ICU patient characteristics in a low income country. BMC Res Notes. 2012; 5: 475. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchievink WI, Wijdicks EF, Parisi JE, et al.: Sudden death from aneurysmal subarachnoid hemorrhage. Neurology. 1995; 45(5): 871–4. PubMed Abstract \| Publisher Full Text\n\nRidenti FA: Neurogenic pulmonary edema: a current literature review. Rev Bras Ter Intensiva. 2012; 24(1): 91–96. PubMed Abstract \| Publisher Full Text\n\nDe La Grandmaison GL, Clairand I, Durigon M: Organ weight in 684 adult autopsies: new tables for a Caucasoid population. Forensic Sci Int. 2001; 119(2): 149–154. PubMed Abstract \| Publisher Full Text\n\nBusl KM, Bleck TP: Neurogenic Pulmonary Edema. Crit Care Med. 2015; 43(8): 1710–1715. PubMed Abstract \| Publisher Full Text\n\nSimon RP: Neurogenic pulmonary edema. Neurol Clin. 1993; 11(2): 309–323. PubMed Abstract\n\nDemyttenaere SV, Nansamba C, Nganwa A, et al.: Injury in Kampala, Uganda: 6 years later. Can J Surg. 2009; 52(5): E146–50. PubMed Abstract \| Free Full Text\n\nJayaraman S, Ozgediz D, Miyamoto J, et al.: Disparities in injury mortality between Uganda and the United States: comparative analysis of a neglected disease. World J Surg. 2011; 35(3): 505–511. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith WS, Matthay MA: Evidence for a hydrostatic mechanism in human neurogenic pulmonary edema. Chest. 1997; 111(5): 1326–1333. PubMed Abstract \| Publisher Full Text\n\nŠedý J, Zicha J, Kuneš J, et al.: Mechanisms of neurogenic pulmonary edema development. Physiol Res. 2008; 57(4): 499–506. PubMed Abstract\n\nWijayatilake DS, Jigajinni SV, Sherren PB: Traumatic brain injury: Physiological targets for clinical practice in the prehospital setting and on the Neuro-ICU. Curr Opin Anaesthesiol. 2015; 28(5): 517–524. PubMed Abstract \| Publisher Full Text\n\nWijayatilake DS, Shepherd SJ: What’s new in the management of traumatic brain injury on neuro ICU? Curr Opin Anaesthesiol. 2014; 27(5): 459–464. PubMed Abstract \| Publisher Full Text\n\nOkello EE, Tumukunde J, Atumanya P, et al.: Dataset 1 in: Prevalence of Neurogenic Pulmonary Edema among patients who died from Head Injury. F1000Research. 2018. Data Source" }	[ { "id": "35040", "date": "11 Jul 2018", "name": "Mabrouk Bahloul", "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 patients with traumatic acute head injury, impaired pulmonary function is a common but poorly understood complication. NPE is a potential early contributor to the pulmonary dysfunction that occurs in patients with head injuries. Although NPE is a frequent complication of traumatic head injury, its occurrence in this specific condition is rarely described. In addition, despite clinical and experimental studies, the mechanisms leading to NPE are not fully understood. As consequence, studying the incidence of NPE after acute head injury is very interesting subject.\nIn this study, in this observational study carried out in Mulago National Referral and Teaching Hospital, Uganda, involving patients who died from head injury. Okello et al had consecutively enrolled subjects into the study upon death over two months (1st June to 31st August 2013) Medical records were reviewed and data on patient demographics and progressive medical/surgical management collected. Particularly, data about diagnosis and diagnostic procedures, medication, fluid administration, ICU admission, mechanical ventilation, and surgical procedures carried out. During the study period, twenty-six patients were enrolled in this study. Autopsy showed features consistent with pulmonary edema in 76.9% of patients, being bilateral in 85% of these. All the 15% in whom edema was unilateral had involvement of the right lung.\n\nThis study suffers from several limitations: 1. There is no hemodynamic exploration for all patients. 2. No electrocardiographic has been reported 3. How pulmonary contusion were excluded? 4. For patients with long bone fractures, how pulmonary fat embolism were excluded? 5. How other causes of pulmonary edema have been excluded: due to severe septic shock! cardiac arrythmia?... 6. Brain CT scan were not performed in all patients\nAll these points represent a several limitations to understand the positive diagnosis and the mechanism of NPE.\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?\nI cannot comment. A qualified statistician is required.\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": [] }, { "id": "36957", "date": "13 Aug 2018", "name": "Roger P. Simon", "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\nOkello et al provide observational data on the incidence of pulmonary edema at autopsy following death from head injury. Consecutive data, of organ weight, with in 24 hours of death, obtained over two months, in 26 patients are provided. Pulmonary edema was documented in 76.8% of patients; weight increase was not found in organs other than the lungs. The mean age was 26 making co morbid cardiovascular factors unlikely.This incidence is similar to that of Simmons (1969) report in fatal military head injuries (pulmonary edema found in 17 of 20 cases). The authors conclusion that neurogenic pulmonary edema begins early and is isolated to the lung adds incidence data to a fatal consequence of head injury about which the pathophysiology and therefore the treatment remains uncertain.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-611
https://f1000research.com/articles/6-1603/v2	20 Sep 17	{ "type": "Research Note", "title": "The first 3D printed multiple sclerosis brain: Towards a 3D era in medicine", "authors": [ "Jagannadha Avasarala", "Todd Pietila", "Todd Pietila" ], "abstract": "Conventional magnetic resonance imaging (MRI) studies depict disease of the human brain in 2D but the reconstruction of a patient’s brain stricken with multiple sclerosis (MS) in 3D using 2D images has not been attempted. Using 3D reconstruction algorithms, we built a 3D printed patient-specific brain model to scale. It is a first of its kind model that depicts the total white matter lesion (WML) load using T2 FLAIR images in an MS patient. The patient images in Digital Imaging and Communications in Medicine (DICOM) format were imported into Mimics inPrint 2.0 (Materialise NV, Leuven, Belgium) a dedicated medical image processing software for the purposes of image segmentation and 3D modeling. The imported axial images were automatically formatted to display coronal and sagittal slices within the software. The imaging study was then segmented into regions and surface rendered to achieve 3D virtual printable files of the desired structures of interest. Rendering brain tumor(s) in 3D has been attempted with the specific intent of extending the options available to a surgeon but no study to our knowledge has attempted to quantify brain disease in MS that has, for all practical purposes, no surgical options.", "keywords": [ "3D printing", "multiple sclerosis", "DICOM files", "image segmentation", "reconstruction algorithms", "patient education", "disease modeling", "neurodegenerative diseases" ], "content": "Introduction\n\nMultiple sclerosis (MS) is a chronic, white and gray matter disease of the central nervous system. Gray matter disease in MS is poorly visualized in conventional MRI but has been increasingly studied in recent years using high strength magnets (de Graaf et al., 2013). The use of MRI in tracking disease of the human brain and spinal cord in patients with MS is central to the diagnosis and treatment of the disease.\n\nDevelopment of computational models for patient-specific requirements based on human pathophysiology individualized to patient-specific data is needed as we move forward with advanced techniques such as 3D printing in medicine. For starters, the potential to improve diagnosis and optimize clinical treatment by predicting outcomes of therapies is attainable. For instance, the accurate prediction of rupture of abdominal aortic aneurysm is possible through patient-based diagnostic tools coupled to medical imaging (Ricotta et al., 2008). However, most results might not apply directly to individual patients yet because they are based on averages (Kent & Hayward, 2007). As an alternative, patient-specific modeling (PSM) can be used as an analytical tool to optimize an individual's therapy. Our study could potentially be useful in building a platform for patient-specific treatment options based on 3D analysis of brain disease, particularly in acute settings such as stroke, mass effect of tumors, midline shift in patients with acute intracerebral hemorrhage, among others.\n\nWith rapid strides made in computer-based technologies, brain atlases are ‘constructed’ by computers. This enables such atlases to become plastic or deformable to fit the size/shape of individual brains. To construct brain atlases, collections of micrographs or schematic drawings of brain sections from one or a few brains are used in which anatomical structures such as nuclei, cortical ribbon or tracts, are identified (Roland & Zilles, 1994). To make assumptions about localization of function and structure at both the macroscopic and microscopic levels, computerized brain atlases are needed. Computerized brain atlases are also used for topographically defined data from the literature (Roland & Zilles, 1994). The spatial resolution is about 1 mm for structural imaging and is below the cellular scale (Roland & Zilles, 1994). For understanding the interaction between brain areas and regions, subcortical nuclei, gyri and sulci, the resolution appears to be sufficient (Toga et al., 2006).\n\nImage segmentation is crucial in medical image analysis and is perhaps the most critical step in many clinical applications (Despotović et al., 2015). In brain MRI analysis, image segmentation is used for measuring and visualizing the brain’s anatomical structures, analyzing changes and identification of pathological regions, as well as for surgical planning and image-guided interventions. Recent advances in brain MRI have provided large amount of data with an increasingly high level of quality but analysis of large and complex MRI datasets is onerous for clinicians, who still extract information manually. Since errors due to inter- or intra-operator variability studies rack up when manual analyses are done, brain MRI data analysis requires inventions in computerized methods to improve disease diagnosis. Increasingly, computerized methods for MR image segmentation, registration, and visualization have been extensively used to assist doctors in qualitative diagnosis (Despotović et al., 2015).\n\nTo help the patient understand the extent of the disease is probably cathartic and revealing although each individual patient may react differently. The primary goal of our endeavor is to educate patient(s) and physician (s) alike regarding the magnitude of a medical disease and the immediacy of treating such a ravaged brain. Our concept borrows from the design and modeling of normal, anatomically-detailed, 3D representations of the normal male and female human bodies and acquisition of transverse CT, MR and cryosection images of representative male and female cadavers in the Visible Human Project.\n\nFrom a patient’s perspective, holding one’s own brain that is built to scale in the palm of a hand delves into a hitherto unknown and previously unexplored dimension. Looking en face at the disease, particularly for a condition that has minimal or no surgical options probably gives patients a better perspective about their disease, but could also evoke fear. With 3D modeling, we enter a novel but untouched world in disease presentation to patients. Only time can tell if more patients embrace such an idea and wish to explore the unknown.\n\n\nData acquisition and segmentation\n\nWe obtained routine MRI images of the brain from a young Caucasian woman in her early 20s who came to our neurology clinic for the first time following a hospital visit for headache, mild gait problems and visual impairment in her right eye that she had developed over the two days prior to presentation. Her MRI images (Phillips 3T TX, software 3.2 version) had the following parameters: Sag T1 SE 5 Thick x 1 gap DWI 5 Thick x 1 gap, Axial FLAIR 5 Thick x 1 gap, Axial T1 SE 5 Thick x 1 gap, Axial PD 5 Thick x 1 gap, Sag FLAIR (reconstructed to Sagittal, Coronal, and Axial 1.0 mm thick x 0 gap, and Sagittal 3D T1 FFE (reconstructed to Sagittal, Coronal, Axial 1.0 mm thick x 0 gap), respectively. The MRI images showed typical white matter lesions that raised concern for MS; her diagnosis was established after ruling out mimics. Since her brain contained an unusually high lesion load, we opted to print a 3D model to fully ascertain the extent of white matter involvement by total lesion volume. We chose T2 FLAIR lesions to compute lesion load and manually identified lesions within each 1 mm slice of the MRI scan in sagittal, coronal and axial planes, respectively. The total combined lesion load was 95,774 mm3, suggesting axonal transection in this volume of brain tissue. A seminal publication (Trapp et al., 1998) showed that active MS lesions, defined on a histological basis, had 11,236 transected axons per mm3 of tissue. This underscores the importance of the burden of disease and the therapeutic challenges that accompany repairing each mm3 of tissue lost to disease. Our patient had a total white matter lesion load of 95,774 mm3 corresponding to a loss of 109 axons. Since no study had characterized a patient’s total lesion volume loss in 3D in MS, comparison of our results to any published literature is not possible.\n\nUsing 3D reconstruction algorithms, we built a highly accurate 3D printed patient-specific brain model to scale. It is a first of its kind that depicts the total white matter lesion (WML) load using T2 FLAIR images in an MS patient. The patient images in Digital Imaging and Communications in Medicine (DICOM) format were imported into Mimics inPrint 2.0 (Materialise NV, Leuven, Belgium) a dedicated medical image processing software for the purposes of image segmentation and 3D modeling. The imported axial images were automatically formatted to display coronal and sagittal slices within the software to aid in the visualization and segmentation process. The imaging study was then segmented into regions and surface rendered to achieve 3D virtual reconstructions in addition to 3D printable files of the desired structures of interest – the brain, ventricles and white matter lesions.\n\nThe cortical surface of the brain was segmented via Thresholding operations which isolates tissue based on gray value in the images corresponding to the cortical brain surface. The ventricles of the brain and lesions were also segmented using Thresholding combined with 3D interpolation to manually refine the accuracy of the segmented regions as shown in Figure 1. After the images were segmented into the defined regions of interest in the images, 3D tessellated surface models were calculated and rendered from the segmented regions (Figure 2). Upon segmentation and reconstruction, accurate brain and lesion volumes can then be calculated.\n\nThe pink represents the total lesion load when amalgamated from all the 3 different slices and planes.\n\nThe digital 3D model of the brain and structures was then virtually sliced on a sagittal plane into its two hemispheres to achieve optimal visualization of the lesions in the eventual 3D printed model. To assist with the utility of the printed model and allow optimal visualization, small holes were created in the mating surfaces of the brain along the sagittal planes to support the insertion of magnets post-3D printing. This enables the brain hemispheres to be separated and then easily assembled using the magnets placed in the corresponding landmarks of each hemisphere. After the completion of the 3D model, STL files of each brain hemisphere were exported for 3D printing on a Connex3 (Stratasys, Eden Prairie, MN, USA) 3D printer. Material-jetting technology was chosen to 3D print the model in order to leverage the need for a combination of transparency and colored regions in the printed models. This technology works by extruding microscopic droplets of curable photopolymer through many jetting heads, building the region one thin layer at a time. The brain cortex was printed using transparent material, with blue representing the ventricles and lesions as depicted in pink (Figure 2).\n\n\nConclusions and future directions\n\nWe emphasize that our model (Figure 3) is primarily educational but can be modified to document the progression or regression of lesions over time. As well, quantification of T1 black hole volume loss, particularly with the development of automated algorithms, is possible (Datta et al., 2006). Hopefully, our work will trigger research into the study of regional/global atrophy, focal/total cortical thickness assessment and deep gray matter changes in 3D, a field that is increasingly coming to light in conventional studies using Structural Image Evaluation Using Normalization of Atrophy software and statistical parametric mapping analysis (Pagani et al., 2005). Additionally, a platform to document changes accurately using computer-assisted automated algorithms that are universally accepted and standardized will be developed. This is critical given the recent EPIC study findings that showed a disappointing trend in how disease-modifying drugs fail to arrest or impact disability in MS patients (Cree et al., 2016) since no drug, if any, affects atrophy measures in a meaningful way. For longitudinal studies, it is crucial that research methods are automated, validated, universally accepted, standardized and based on computer-based image analysis tools that can sift through large data sets. Additional enhancements for our 3D model could include such innovations as Cold Spring Harbor’s G2C interactive normal brain models, funded by the Dana Foundation and Hewlett Foundation, wherein structure/function relationships can be gleaned when a 3D brain with disease is superimposed on an interactive normal 3D brain model giving patients and physicians a new perspective on how different anatomical structures are involved and affected in health and disease. Since no two patients are similar, scan quality can vary but so do their file formats. Yet, if the end goal is improvement in quality patient care, one would want to ensure that the 3D models accurately represent the patient’s anatomy which is what one would expect as 3D technologies continue to evolve.\n\nVentricles are shown in blue and white matter lesions are depicted in pink.\n\nMany automated segmentation methods that detect brain lesions have been developed in MS (Udupa et al., 2001; Wu et al., 2006; Zijdenbos et al., 2002) but no study has been validated for commercial or routine use, nor has the depiction of the impact of lesion load in a 3D printed model been published. If such technology can be developed and transferred to the ICU settings, medical and surgical decisions could perhaps be handled better, particularly in acute neurological disorders that cause rapid clinical changes and worsening mass effect and midline shift following intracerebral bleeding, hydrocephalus or cerebral edema owing to mass effect of tumors. New guidelines could be developed for therapeutic and surgical interventions. Could 3D printing introduce a new angle to how lesion load is defined? Can one visualize 3D printing becoming a teaching, diagnostic and decision-making tool in the ICU setting? We think that to accurately document changes that occur in acute neurological diseases such as hemorrhagic strokes with or without mass effect, or cerebral edema from varied causes, a 3D model would be ideal if not mandatory, particularly if available in real time for decision-making in treatment options and patient education.\n\nSince no radiological markers accurately quantify disability in MS, how does one assess objectively, the effect of disease modifying drugs on MS outcomes research? As technology evolves, a routine CT and MRI scan can probably be converted instantly into a 3D model with the help of automatic segmentation algorithms that could be used to document volumetric changes both global, regional and deep gray matter structures. We hope our study is the first step towards such a goal.\n\nQuantitative analysis of WML in large clinical trials assumes a major role particularly in cerebrovascular disease, diabetes mellitus and Alzheimer’s disease, wherein 30% of patients could have some degree of vascular pathology. In population studies, such as the Cardiovascular Health Study (CHS) or the Rotterdam Scan Study (RSS) WMLs have been shown to be associated with age, clinically silent stroke, higher systolic blood pressure, hypertension, atrial fibrillation, among others (de Groot et al., 2000; de Groot et al., 2000; Longstreth et al., 1996). An urgent unmet need is the assessment of MRI data of WML load in various disease states that is standardized, automated and followed longitudinally. Hopefully, this study is a first of many such attempts in that evolutionary path moving forward.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of the patient’s details and accompanying images.\n\n\nData and software availability\n\nThe MRI files underlying the 3D model of this patient’s brain have not been included to maintain patient anonymity.\n\nAlternative software packages that are available include Slicer (open source) or Osirix (free demo available) to segment the imaging data, and Meshmixer (open source), a digital CAD software, to prepare the 3D model for printing.", "appendix": "Author contributions\n\n\n\nJA: Concept, data collection, MRI analysis, manuscript preparation; TP: 3D printing, presentation and development of the model, MRI data extraction from DICOM files.\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 Movie 1: Video of 3D brain with multiple sclerosis.\n\nClick here to access the data.\n\n\nReferences\n\nDatta S, Sajja BS, He R, et al.: Segmentation and quantification of black holes in multiple sclerosis. NeuroImage. 2006; 29(2): 467–474. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Graaf WL, Kilsdonk ID, Lopez-Soriano A, et al.: Clinical application of multi-contrast 7-T MR imaging in multiple sclerosis: increased lesion detection compared to 3 T confined to grey matter. Eur Radiol. 2013; 23(2): 528–540. PubMed Abstract \| Publisher Full Text\n\nde Groot JC, de Leeuw FE, Oudkerk M, et al.: Cerebral white matter lesions and cognitive function: the Rotterdam Scan Study. Ann Neurol. 2000; 47(2): 145–151. PubMed Abstract \| Publisher Full Text\n\nde Groot JC, de Leeuw FE, Oudkerk M, et al.: Cerebral white matter lesions and depressive symptoms in elderly adults. Arch Gen Psych. 2000; 57(11): 1071–1076. PubMed Abstract \| Publisher Full Text\n\nDespotović I, Goossens B, Philips W: MRI segmentation of the human brain: challenges, methods, and applications. Comput Math Methods Med. 2015; 2015: 450341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKent DM, Hayward RA: Limitations of applying summary results of clinical trials to individual patients: the need for risk stratification. JAMA. 2007; 298(10): 1209–1212. PubMed Abstract \| Publisher Full Text\n\nLongstreth WT Jr, Manolio TA, Arnold A, et al.: Clinical correlates of white matter findings on cranial magnetic resonance imaging of 3301 elderly people. The Cardiovascular Health Study. Stroke. 1996; 27(8): 1274–1282. PubMed Abstract \| Publisher Full Text\n\nPagani E, Rocca MA, Gallo A, et al.: Regional brain atrophy evolves differently in patients with multiple sclerosis according to clinical phenotype. AJNR Am J Neuroradiol. 2005; 26(2): 341–346. PubMed Abstract\n\nRicotta JJ, Pagan J, Xenos M, et al.: Cardiovascular disease management: the need for better diagnostics. Med Biol Eng Comput. 2008; 46(11): 1059–1068. PubMed Abstract \| Publisher Full Text\n\nRoland PE, Zilles K: Brain atlases--a new research tool. Trends Neurosci. 1994; 17(11): 458–467. PubMed Abstract \| Publisher Full Text\n\nToga AW, Thompson PM, Mori S, et al.: Towards multimodal atlases of the human brain. Nat Rev Neurosci. 2006; 7(12): 952–966. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrapp BD, Peterson J, Ransohoff RM, et al.: Axonal transection in the lesions of multiple sclerosis. N Engl J Med. 1998; 338(5): 278–285. PubMed Abstract \| Publisher Full Text\n\nUdupa JK, Nyúl LG, Ge Y, et al.: Multiprotocol MR image segmentation in multiple sclerosis: experience with over 1,000 studies. Acad Radiol. 2001; 8(11): 1116–1126. PubMed Abstract \| Publisher Full Text\n\nUniversity of California, San Francisco MS-EPIC Team, Cree BA, et al.: Long-term evolution of multiple sclerosis disability in the treatment era. Ann Neurol. 2016; 80(4): 499–510. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu Y, Warfield SK, Tan IL, et al.: Automated segmentation of multiple sclerosis lesion subtypes with multichannel MRI. NeuroImage. 2006; 32(3): 1205–1215. PubMed Abstract \| Publisher Full Text\n\nZijdenbos AP, Forghani R, Evans AC: Automatic \"pipeline\" analysis of 3-D MRI data for clinical trials: application to multiple sclerosis. IEEE Trans Med Image. 2002; 21(10): 1280–1291. PubMed Abstract \| Publisher Full Text" }	[ { "id": "26575", "date": "16 Oct 2017", "name": "Daniel S. Reich", "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 article demonstrates the creation of a 3D printed model of the brain of a multiple sclerosis patient, allowing for enhanced visualization of white matter lesions. This technique may be useful for medical education and may provide patients with a more tangible representation of their disease pathology. By utilizing clinically relevant MRI sequences and an imaging segmentation tool designed for a clinical workflow, the authors present a method that can be implemented by clinicians at other institutions. However, the article has several weaknesses that must be addressed:\nThe authors report that the patient provided written consent for publication of his/her images, however there is no information about whether the study was approved by the local IRB, or, instead, a waiver was received. IRB approval, or a waiver of IRB approval, is required, by law, prior to commencing a medical research study.\n\nThe paper is classified as a “Research Note,” which, per the F1000Research guidelines, “include single-finding papers that can be reported with one or two illustrations (figures/tables), descriptions of unexpected observations, and lab protocols. Posters from conferences or internal meetings may be summarized as Research Notes. In many cases, some additional detail, particularly in the methods, description of the results, and/or discussion/conclusions will be required to make sure that readers (and referees) have enough information to understand the description of the work.” There is no research in this paper; rather, it is an educational demonstration, as the authors themselves report. In fact, it is not clear that this report falls under any of the F1000Reserach article categories.\n\nThe patient’s diagnosis of multiple sclerosis (MS) is not entirely clear based on the presented information. As it is noted that the patient has an unusually large lesion load (with bilateral temporal lobe symmetry, per Figures 1, 2, and Supplementary Movie 1), we would ask that the authors clarify if the patient met the 2010 McDonald criteria (Polman et al., 2011), specifically dissemination in time (DIT). Further, if DIT was apparent based on the hospital visit MRI as compared to the images collected at the neurology clinic, printing a 3D model for each of these time points would strengthen the author's argument that the brain model could be used to demonstrate disease progression. But in that case, they would need to quantify the change using their approach. We also ask that the authors clarify the clinical measures used to reach the diagnosis of MS for this patient.\n\nThe authors state that “Since no study had characterized a patient’s total lesion volume loss in 3D in MS, comparison of our results to any published literature is not possible.” This is clearly not the case. Indeed, the segmentation in this paper was done on 2D slices of the 3D image volumes, and that is routine in the field.\n\nThe discussion of axonal transections, based on data from Trapp et al., 1998, is bizarre. As the authors themselves note, no two MS cases are identical, and therefore precise estimates of the number of transected axons in the current patient’s brain are not justified. Moreover, the authors provide no information about how they determined whether lesions in their patient’s brain were active or chronic, which is highly relevant in this regard. This entire section should be removed.\n\nThe authors should discuss a recent publication relevant to this article: Newton, Braeden D., et al. \"Three‐Dimensional Shape and Surface Features Distinguish Multiple Sclerosis Lesions from Nonspecific White Matter Disease.\" Journal of Neuroimaging (2017).\n\nWhile the authors emphasize the utility of automated/computational algorithms in MRI in both the introduction and the discussion, the method presented here is based entirely on manual segmentation and thresholding. Given this emphasis, we ask that the authors explain why they chose not to use an automated method for segmentation of lesions, such as those presented in Carass et al., 2017.\n\nThe authors suggest that their work could stimulate research into cortical atrophy. We ask that the authors comment on the extent of atrophy required to notice an appreciable change in a 3D model and how that might compare with the known rate of cortical atrophy in MS literature.\n\nThe authors state that they believe a 3D printed model may become mandatory as a decision-making tool in some acute neurological diseases if provided in real time. Given the assumption that segmentation would be automated, we ask that the authors provide the approximate printing time and cost of the 3D model to provide context of the practicality of this use.\n\nThe references – particularly the ones that deal with segmentation of MS MRI scans – are out-of-date.\n\nAs a general point, the article is written in a very non-conventional format, and there are many extraneous details and a good bit of philosophizing, which really has no place in a scientific article.\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? 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? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "3114", "date": "17 Oct 2017", "name": "Jagannadha Avasarala", "role": "Author Response", "response": "The authors thank the reviewer for his erudite observations. Our responses are as follows1. IRB protocol was not a requirement at our institution (Avasarala J) but we are obtaining one now, to meet compliance standards. It is not our intent to do anything that is outside the rules of our institution.2. Research note or technical note is, in theory, semantics. Software application to DICOM files was done as noted (Todd, P) in the paper. I am not sure that anything specific is being missing, but MRI data transformation was done, into DICOM files and data extracted into the software suite as described. We can expand on the technicalities of the software application, if need be.3. The diagnosis was established based on T1 gad enhancing lesions AND T2 lesions present at the original time of diagnosis in the hospital (DIT and DIS) AND clinical examination AND clinical presentation AND exclusion of other mimcs as is the standard of care at our institution. The principal author is MS-fellowship trained (Washington U School of Medicine, 2000-03, St Louis, MO) and made the diagnosis based on evidence, clinical/radiological findings.4. Segmentation in 2D is a very old technique but NO reconstruction of 3D volume losses in a 3D PRINTED model of human brain with MS was ever done or published, hitherto. There is no circumstance described in the literature that we could find that describes a physician presenting to his/her patient with a 3D brain (of the patient) with a goal of educating the patient (instead of showing lesions on a flat, 2D screen). Segmentation of MRI lesions is not new but collation of 2D data into a 3D model that is printable, is.5 We disagree. This patient presented with de novo MS. The lesions, as described, are T2 lesions and were volumetrically assessed. Prior to the publication, one of us (JA) wrote to Dr Trapp and he agreed that ANY new T2 lesion or T1 enchanced lesion would represent a 'new lesion' and as noted, the patient was presenting for the first time. These lesions are not chronic, they are acute. Any clinical trial of any phase 3 drug trial for MS drugs notes two fundamental aspects of a new lesion definition on MRI - and they are a) T1-gad lesions and b) NEW T2 lesions. Using that definition and since the patient presented de novo, these are acute lesions. Acute lesion volumetric analysis (Trapp et al) shows that I cu mm3 destroys 11 K axons, from axonal transection. Unless this fundamental finding is being questioned, the axonal loss from the total volumetric loss in our patient corresponded to the number of axons transected, as noted. This section will not removed and is the core of this manuscript.6 and 7. We can consider these suggestions. As for segmentation, there is NOT one standardized approach for ANY disease of the CNS, including those that claim automation, that everyone agrees on, or is a gold-standard. Manual automation does not disqualify the study, per se. Automated versions are fundamentally developed to look at massive amounts of data but to date, there is not even a single database in the world of MS that has defined brain atrophy using automated segmentation techniques. As well, no findings across multiple samples from various available datasets via normalization are published - some publications claim these findings, but again, no standard format exists. Therefore, no normalization techniques are available although numerous published data claim that one is better than the other.8-11. There is no philosphical musing in our manuscript. Our references are what we felt were appropriate and relevant for the subject being discussed. As for points 8 and 9, they are well received and we can provide additional data on cost and time for using similar technology as it stands today." } ] }, { "id": "26539", "date": "30 Oct 2017", "name": "Toshihiro Mashiko", "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\n[Overall comments] The authors have described an extremely interesting three-dimensional (3D) model for MS, with precise segmentation of the MS lesion. However, the usefulness and applicability of this model is unclear. Although the authors have mentioned various points in the “Conclusions and Future Directions section,” they have not emphasized on the necessity to use 3D models in their study. Research is to discover the unknown. However, in this paper, it has not been thoroughly achieved. The authors must initially apply a suitable research design to demonstrate the usefulness of a 3D model for MS. For “Future Directions,” the basis shall be stated in each item.\n[Is the work clearly and accurately presented and does it cite the current literature?] The method of fabrication of the model is clearly and accurately presented. However, the purpose and results of this study are unclear. In addition, the authors have stated that the 3D models assist in increasing patient awareness and knowledge without providing substantial evidence. This study has essentially succeeded in fabricating a 3D model for MS; however, there is no new knowledge.\n[Is the study design appropriate and is the work technically sound?] There is no description regarding the study design.\n\n[Are sufficient details of methods and analysis provided to allow replication by others?] No analysis has been conducted.\n\n[Are all the source data underlying the results available to ensure full reproducibility?] The results are not described.\n\n[Are the conclusions drawn adequately supported by the results?] Because the results are not shown, the conclusion cannot be evaluated.\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? 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? No", "responses": [ { "c_id": "3146", "date": "01 Nov 2017", "name": "Jagannadha Avasarala", "role": "Author Response", "response": "The authors thank Toshihiro Mashiko for his comments.Our responses are The single most important aspect(s) of our paper is that a) feasibility of printing a 3D brain with disease is do-able, and b) that the applicability of such technique(s) can be extended to the ICU provided progress in technology developments are attained. The 3D technology applications in the SURGICAL field are nothing new and are, in fact, routine - 3D printing techniques are practical and anatomically accurate methods of producing patient‑specific models for surgical planning, simulation and training, tissue‑engineered implants, and secondary devices is well described and almost universally applied in select cases, probably well known to Dr Mashiko. Our idea is extend this thought process to the MEDICAL field, and in particular, neurology, as an example. The purpose of this study and results are presented to demonstrate feasibility of the above 'idea'. All it takes is to utilize the capabilities of companies that can turn 2D data such as MRI images into definable, patient-education-friendly models in 3D that can augment what is conventionally being presented the world over in a 2D data format." } ] }, { "id": "26540", "date": "27 Dec 2017", "name": "Ramin Javan", "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 article I believe is better suited as a “technical note” rather than a “research note” as it predominantly describes the methodology of creating a novel 3D printed model along with discussion of possible or presumed future implications and uses. There is no comparison, no survey data, no statistical data or analysis, or any p-values that ate pertinent in this paper for it to be a research note. That being said, the model is extremely visually appealing and can certainly be used for patient counseling, trainee education and interdisciplinary decision making.\n\nThe underlying technique though may be applied in many other applications where an internal pathology is to be demonstrated within a normal structure or an organ, with many publications describing it either using Polyjet technology or through stereolithography with transparent resin. With respect to multiple sclerosis, however, the vast majority of cases do not have such a heavy load of white matter disease as demonstrated here, except for cases of primary progressive MS or advanced MS. So this model, exaggerates the possible use of 3D printing in MS, as authors themselves describe the case presentation to have an unusually high load of white matter disease.\nAnother consideration is the cost of the model (which would help readers if it was described either ordered through Materialise or the cost of the Connex printer and material) and whether it adds benefit for that cost and the time needed for segmentation and 3D printing. However, as authors mention, this can and will likely change in the future both in terms of automation of segmentation as well as the cost.\n\nOne consideration to take into account to improve upon the current model would be to also add another color mask that shows enhancement of lesions demonstrating degree of active disease (especially with newer multicolor Polyjet 3D printers that can print up to 6 non-support material colors). This may help clinicians in the acute setting determining the severity, extent or percentage of plaques involved in active demyelination. It can also be helpful in determining predominance in certain areas of the brain (as mentioned by authors) or for comparing to prior white matter lesion load or comparing with other previous acute presentations.\n\nWith respect to the authors’ statement regarding the need for automated quantitative analysis of white matter disease, the specific role of 3D printings is questionable while the actual 3D volumetric measurement is of the essence, readily provided by the software. A 3d model may help clinicians determine whether the progression has occurred predominantly in a certain region of the brain for example. This, however, can also be done through virtual 3D visualization but also with the use of software calculations, probably even more accurately, if desired.\n\nLastly, financial disclosure must be detailed under “Competing Interest” since second author is an employee of Materialise.\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? 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? No", "responses": [] } ]	2	https://f1000research.com/articles/6-1603
https://f1000research.com/articles/7-222/v1	23 Feb 18	{ "type": "Research Note", "title": "Xylariales: First results of mycological exploration in the Sangay and Llanganates National Park, Ecuador", "authors": [ "María-Fernanda Guevara", "Paula Salazar", "Bence Mátyás", "María-Eugenia Ordoñez", "María-Fernanda Guevara", "Paula Salazar", "María-Eugenia Ordoñez" ], "abstract": "In the present study fungi collections were sampled in the Sangay (SP) and Llanganates (LP) National Parks, from which sequences of the regions of the internal transcribable spacer (ITS1-5.8S-ITS2) of the ribosomal DNA were obtained (RDNA). The taxonomic identification of fungi of the order Xylariales was achieved with the bioinformatic tools, to further study the phylogenetic relationships among the collected individuals and thus contribute with base information on their biological diversity, necessary to design and implement measures for the conservation of fungi. All records belong to the genus Xylaria, of these eight belong to PL and two to SP. A record was not identified at the species level, suggesting that it could be a new species. A phylogenetic tree of Maximum Likelihood was built.", "keywords": [ "Diversity", "ITS", "Llanganates", "National Park", "Sangay", "Xylarial" ], "content": "Introduction\n\nSangay (SP) and Llanganates (LP) National Parks in Ecuador are considered as high priority conservation units in the Tropical Andes, due to their high biodiversity and high endemism1,2. However, their mycological diversity is still unknown. This study aims to contribute to the conservation of fungi, showing the results of their diversity, based on molecular taxonomy, by analyzing the ITS (internal transcribed spacer) regions. ITS is the accepted ’barcode’ for fungi3. For this, the DNA sequence of specimens of exploratory fungal collections were analyzed within the aforementioned parks. Here we present results exclusively for the Xylariales order.\n\n\nMethods\n\nSample collection was carried out during the months of January and February 2015. The fruiting bodies collected were deposited in the QCAM Fungarium (Catholic University Mycology Collection, Quito). Table 1 displays the collection codes, as stored at the QCAM. The ITS1-5.8S-ITS2 region was amplified by PCR with primers (provided by Invitrogen Co., Carlsbad, CA, USA) ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’)4 and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’)5. The amplified fragments were sent for sequencing to Macrogen Inc. (Seoul, South Korea). The obtained sequences were edited in Geneious R8 (Biomatter Ltd. 2005–2012) by selecting the “de novo assemble” tool and then trimming the ends. The consensus sequence was manually edited, and submitted to GenBank. Sequence data were analyzed by comparison with sequences available in GenBank. An assignment to the lower taxonomic level was made by direct homology of the consensus sequences with the search results in BLASTn (NCBI) optimized for highly similar sequences (megablast), alignments that presented 100\n\nSequence data were aligned with Geneious R8 and later manually adjusted with Mesquite version 3.046. Public sequences are available in GenBank that corresponded to specimens that gave the greatest homology in BLASTn with the sequences of the collected specimens were included in the analyses. Phylogenetic trees were constructed in Geneious R8 using the PhyML7 plugin for Maximum Likelihood (ML) with a Custom (010230) substitution model determined by jModelTest 2.1.4.8,9, according to Corrected Akaike Information Criterion (AICc)10,11. A bootstrap of 1000 replicas was used.\n\n\nResults\n\nAll the specimens collected were of the genus Xylaria. The eight specimens from Llanganates National Park were identified as X. enterogena, X. fissilis, X. schweinitzii, X. telfairii and three unidentified species. For the two samples from Sangay National Park, one was X. telfairi and the other was an unidentified species. Differences in the number of samples found at each park could be due to the sampling effort that was different in each park, however, this is a sample of the high biodiversity in LP and SP. The unidentified species were different in each park. The analysis shows that there are no shared species of Xylaria at the two parks (Table 1), this is important for conservation decisions. The phylogenetic relationships recovered from the analysis of the ITS sequences (Figure 1) shows two major groups. The first major group, composed by clades A and B, is well supported (bootstrap > 95) includes specimens from LP and PS. Clade A includes all X. entogena specimens and is well supported (bootstrap > 95). Clade B includes all X. telfairii specimens and Xylarya sp.1 specimens (bootstrap < 50), it could be supposed that Xylarya sp.1 belongs to the X. telfairii species, but because the high difference among the sequences it is considered a different species. In the second major group (bootstrap > 75), clade C is sister to clades D, E and F; this major group includes specimens from LP and SP, too. Clade C includes all X. schweinitzii specimens (bootstrap > 95). Clade D (bootstrap > 80) includes Xylaria sp. 2, the closest sequence to Xylaria sp. 2 from SP was a previously reported collection also from Ecuador12 in a cloud forest in the province of Imbabura, that was also identified only at the genus level. Clades E (bootstrap > 95) shows Xylaria sp. 3, the closest sequence to this individuals belongs to the same previously reported collection12, identified only at the genus level. Clade F (bootstrap = 100) includes Xylaria fissilis sequences from LP and one from 12; clade F also includes Xylaria sp. 4 an unidentified specimen.\n\nThese unidentified specimens might represent new species. Additional loci and more detailed morphological analyses are needed to determine this. The genus Xylaria is probably the largest in the family Xylariaceae, with 35 estimated genera13, but the real number remains unknown14. Studies in relation to the biological diversity of this order in the National Parks of Ecuador are scarce, more systematic field studies would surely reveal a greater diversity of families, genera and species within the Xylariales in SP and LP, as well as other regions and protected areas of Ecuador, especially if we take into account the cosmopolitan distribution of Xylaria13. In fact, new fungal species in SP, belonging to the Agaricales, have recently been described15.\n\n\nConclusions\n\nThe results obtained allow us to establish a baseline for the biological diversity of the Xylariales in SP and LP, an important step to the conservation of fungi. This is the main contribution of this study. We found four species of Xylaria: X. enterogena, X. telfairii, X. schweinitzii, and X. fissilis, and four potential new species; the species found in LP are different from those found in SP. However, there is much more to discover. A huge and complex task is pending. To advance our understanding of this Kingdom we must start by deciphering the diversity of fungi present in these sites.\n\n\nData availability\n\nThe sequencing data are available on the NCBI Genbank webpage:\n\nXylaria enterogena: https://www.ncbi.nlm.nih.gov/nuccore/MG768840\n\nhttps://www.ncbi.nlm.nih.gov/nuccore/MG768839\n\nXylaria fissilis: https://www.ncbi.nlm.nih.gov/nuccore/MG768834\n\nXylaria schweinitzii: https://www.ncbi.nlm.nih.gov/nuccore/MG768836\n\nXylaria telfairii: https://www.ncbi.nlm.nih.gov/nuccore/MG768832\n\nXylaria sp. 1: https://www.ncbi.nlm.nih.gov/nuccore/MG768838\n\nhttps://www.ncbi.nlm.nih.gov/nuccore/MG768841\n\nXylaria sp. 2: https://www.ncbi.nlm.nih.gov/nuccore/MG768833\n\nXylaria sp. 3: https://www.ncbi.nlm.nih.gov/nuccore/MG768837\n\nXylaria sp. 4: https://www.ncbi.nlm.nih.gov/nuccore/MG768835", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe Secretaria de Educación Superior, Ciencia, Tecnología e Innovación del Ecuador (SENESCYT), Arca de Noé Initiative and the QCAM Fungarium supported the project.\n\n\nAcknowledgements\n\nWe are grateful to Marcel A. Caminer, Charles W. Barnes and Cristina E. Toapanta for their invaluable contribution to the development of the present research.\n\n\nReferences\n\nBajaña F, Rivas J, Sánchez D, et al.: Informe de la evaluación inicial del Parque Nacional Sangay como Sitio de Patrimonio Natural de la Humanidad. Quito-Ecuador: Ministerio del Ambiente, Fundación Natura, EcoCiencia y UICN-Sur. 2002.\n\nMinisterio del Ambiente del Ecuador: Plan de Manejo Parque Nacional Llanganates. Quito-Ecuador, 2013. Reference Source\n\nSchoch CL, Robbertse B, Roberts V, et al.: Finding needles in haystacks: linking scientific names, reference specimens and molecular data for Fungi. Database (Oxford). 2014; 2014; pii: bau061. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGardes M, Bruns TD: ITS primers with enhanced specificity for basidiomycetes--application to the identification of mycorrhizae and rusts. Mol Ecol. 1993; 2(2): 113–118. PubMed Abstract \| Publisher Full Text\n\nWhite TJ, Bruns T, Taylor J: Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics. Mycologia. 2013; 64(1). Reference Source\n\nMaddison WP, Maddison DR: Mesquite: a modular system for evolutionary analysis. Version 3.04. 2015. Reference Source\n\nGuindon S, Dufayard JF, Lefort V, et al.: New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010; 59(3): 307–321. PubMed Abstract \| Publisher Full Text\n\nDarriba D, Taboada GL, Doallo R, et al.: jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9(8): 772. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2013; 52(5): 696–704. PubMed Abstract \| Publisher Full Text\n\nAkaike H: Information theory and an extension of the maximum likelihood principle. In: Petrov BN, Csaki F. (Eds.), 2nd International Symposium on Information Theory. Budapest: Akademiai Kiado, Budapest, Hungary, 1973; 267–281. Reference Source\n\nPosada D: jModelTest: phylogenetic model averaging. Mol Biol Evol. 2008; 25(7): 1253–1256. PubMed Abstract \| Publisher Full Text\n\nThomas DC, Vandegrift R, Ludden A, et al.: Spatial Ecology of the Fungal Genus Xylaria in a Tropical Cloud Forest. Biotropica. 2016; 48(3): 381–393. Publisher Full Text\n\nLee JS, Ko KS, Jung HS: Phylogenetic analysis of Xylaria based on nuclear ribosomal ITS1-5. 8S-ITS2 sequences. FEMS Microbiol Lett. 2000; 187(1): 89–93. PubMed Abstract \| Publisher Full Text\n\nHsieh HM, Lin CR, Fang MJ, et al.: Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily. Mol Phylogenet Evol. 2010; 54(3): 957–969. PubMed Abstract \| Publisher Full Text\n\nCrous PW, Wingfield MJ, Burgess TI, et al.: Fungal Planet description sheets: 558–624. Persoonia. 2017; 38(1): 240–384. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "31232", "date": "01 Mar 2018", "name": "Francisco J. Flores", "expertise": [ "Reviewer Expertise Phylogenetics" ], "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 \"Xylariales: First results of mycological exploration in the Sangay and Llanganates National Park, Ecuador \" provides internal transcribed spacer sequences from Xylaria isolates collected from Ecuadorian national parks. Information about fungal diversity in Ecuador is still scarce, which makes this manuscript relevant. Nevertheless, I have several concerns, mainly:\nPhylogenetic analysis using only the ITS sequence is very superficial, and, most likely, it won´t show an accurate representation of the real evolutionary relationships between isolates. There is no mention about the morphological characteristics of isolates. Is the morphology congruent with the genotype?\nThe manuscript needs major revision to improve clarity.\nSeveral suggestions are made in the file linked below. https://f1000researchdata.s3.amazonaws.com/linked/196501.Bence_Matyas_rev.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? 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": [] }, { "id": "32603", "date": "19 Apr 2018", "name": "D. Jean Lodge", "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\nIntroduction. Because the aim is to compare these two parks in Ecuador, it is important that the elevation ranges of each are included, as well as the distance between the two parks.\n\nMethods, p. 3, last line seems to be an incomplete sentence. It ends with ‘BLASTn’ (NCBI) optimized for highly similar sequences (megablast), alignments that presented 100’ It is probably meant to say ‘overlapped 100%.’\n\nResults: Text for Clade E does not match the phylogram. The text says that sp. 3 was related to another collection identified only at the genus level. However, the phylogram shows a well-supported clade (98% BS) comprised of sp. 3 and X. curta KP133352 ECU. I would expect X. curta to be reasonably common there. There is either an error in identification of KP133352 ECU or the text in resutls needs to be changed, and the abstract and first line of results and conclusions also need to be changed to include X. curta.\n\nThat also means that the unidentified species need to be renumbered in the text, table, and phylogram, with sp. 4 becoming sp. 3.\n\nThere are likely 3 undescribed species included in this study. The journal allows for photographs, and photos should be added to the manuscript. Even if photos were not taken of the fresh specimens, photos of the dried specimens can be used, and are very helpful. If photos of the asci, especially the ascus plug stained with iodine/Melzer’s reagent, and photos of the ascospores showing the germ slits, that would be very helpful also for future work. The ascospore length and width ranges, the shape and extent of the germ slit, and the shape and size of the ascus plug would be helpful additions.\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? 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/7-222
https://f1000research.com/articles/7-206/v1	19 Feb 18	{ "type": "Research Article", "title": "Evaluation of defense strategy against Influenza A in cell line models", "authors": [ "Ekaterina Antonova", "Olga Glazova", "Anna Gaponova", "Aykaz Eremyan", "Natalya Grebenkina", "Svetlana Zvereva", "Natalya Volkova", "Pavel Volchkov", "Olga Glazova", "Anna Gaponova", "Aykaz Eremyan", "Natalya Grebenkina", "Svetlana Zvereva", "Natalya Volkova" ], "abstract": "Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 virus is getting increasingly infective for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild aquatic birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase (hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS.\n\nResults: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialilation to prevent infection. Tissue specific expression of the sialidases could protect domestic birds from infection.", "keywords": [ "sialidases", "Influenza A", "defence strategy", "exogenous expression", "α(2", "3)-sialylation" ], "content": "Introduction\n\nThe global influenza pandemic remains a real threat. Avian influenza viruses H5N1 have killed millions of birds, including domestic poultry, causing enormous financial losses. Several thousands of cases of human infection were recorded and some with a fatal outcome (see World Health Organisation (WHO) assessment of Avian Flu pandemic threat). The risk of a new pandemic of viral infection remains high. Seasonal flu vaccination is used as a traditional way to prevent the infection spread, however it has a lot of limitations. Therefore, it is critical to develop alternative approaches to prevent influenza infection. An alternative way to reduce the infection could be the creation of genetically modified domestic birds. It will decrease the risk of the infection spread, because domestic birds are known to transmit the infection acting as an intermediary between wild ducks to humans (Kim et al., 2009). It is necessary to select gene-candidates for developing the approach.\n\nThe best way to prevent the infection is to impede the virus from entering the cell. Influenza virus hemagglutinin (HA) provides attachment to the host cell that leads to fusion between the virion envelope and the host cell membrane (Skehel & Wiley, 2000). Surface sialic acid (SA) residues are host cell epitopes that are recognized by influenza virus A and B (Ito, 2000). SA are a family of nine-carbon acidic monosaccharides that naturally terminate sugar chains attached to the proteins on the cell surface (Varki et al., 2009). Neuraminidase (NA) - the second major surface antigen - is an exoglycosidase, or saildase, that cleaves SA from cell membrane glycolipids and glycoproteins, thus destroying the recognition epitopes on the surface of the host cell for the viral receptor HA. NA activity helps viral particles penetrate through mucous secretions that are rich in sialic acids, to reach the target cells in the airway epithelium (Palese et al., 1974). The role of the enzyme in facilitating release of newly formed viral particles from the infected cell surface and preventing aggregation of the viral particles was experimentally confirmed (Matrosovich et al., 2004; Palese et al., 1974). There are known antiviral agents - oseltamivir and zanamivir - that inhibit NA, blocking the release of virus particles from infected cells (Moscona, 2005; Varghese, 1999). Sialidases have also demonstrated to be effective inhibitors of Influenza virus infection in vitro. It has been shown that cells treated with Vibrio cholerae or Micromonospora viridifaciens bacterial sialidase are resistant to influenza virus infection (Air & Laver, 1995; Bergelson et al., 1982; Griffin et al., 1983; Stray et al., 2000).\n\nWe focused on exogenous expression of sialidases as a defense strategy against influenza infection (Figure 1). Here we show the protective effect of exogenous expression of different sialidases. The range of exogenous sialidases has diverse activity and specificity towards sialic acid residues. Tissue-specific expression of sialidases in transgenic poultry might protect domestic birds against Influenza virus.\n\nInfluenza A virus penetrates into the host cell through the binding with the host cell surface sialic acids. (B) Removal of sialic acids through exogenous sialidase expression protects cells from viral infection.\n\n\nMethods\n\nViral neuraminidase. Viral neuraminidase from human Influenza A virus was amplified from the plasmid pNAHA (Plasmid # 44169; Addgene, Cambridge, MA, USA) that encodes the neuraminidase gene of Influenza A / Puerto Rico / 8/34, subtype N1. The amplified product was inserted by restriction cloning in the lentiviral transfer plasmid based on a pHAGE backbone (#24526, Addgene) after the CMV promoter. The RFP reporter was cloned in the same reading frame and the resulted construction was pHAGE-CMV-infNA-P2A-RFP (Supplementary Figure 1A).\n\nSialidases from Salmonella typhimurium and Actinomyces viscosus. Synthesis of the codon-optimized bacterial sialidase from Salmonella typhimurium (Uniprot / P29768, 2-382aa) and the codon-optimized bacterial sialidase from Actinomyces viscosus (uniprot / Q59164, 347-631aa) fused with the transmembrane domain of viral neuraminidase (1 - 75 aa) were done in BioCat. The transmembrane domain was added to provide membrane localisation of the enzymes, since sialidase Salmonella.t. and A. viscosus are cytoplasmic. The codon-optimization was done with the IDT tool with the choice of the genome of Gallus gallus. The genes were ordered in the standard vector pUC57 and were flanked by the restriction sites NheI and SphI for S. typhimurium and XhoI and SphI sites for A. viscosus. The genes were excised from the vector pUC57 using the sites and inserted into the pHAGE backbone after the the CMV promoter. As a result, the plasmids pHAGE-CMV-Sal.t.Sia-P2A-RFP and pHAGE-CMV-Act.v.Sia-P2A-RFP have been cloned (Supplementary Figure 1A).\n\nThe genes coding sialidase 3 (membrane sialidase) in G.gallus and H.sapiens are homologous based on the analysis in HomoloGene (NCBI). We selected human membrane sialidase (Gene ID: 10825) for cloning because there is more information about it compared with other orthologs. The coding exons were amplified from human genomic DNA and overlapped. The resulted product was inserted by restriction cloning in the lentiviral transfer plasmid based on the pHAGE backbone (#24526, Addgene) after CMV promoter. The RFP reporter was cloned in the same reading frame and the resulted construction was pHAGE-CMV-hNeu3-P2A-RFP (Supplementary Figure 1A).\n\nEnzymatically inactive neuraminidase was taken as a negative control. Two amino acids at positions 262-263 (EE) of the Influenza A virus strain A/Puerto Rico/8/1934 H1N1 are responsible for the substrate binding. The inactive variant can be obtained by the mutation E262D (Huang et al., 2008). The mutation was made by two overlapping primers: 5’-GCACCTAATTCTCACTATGAtGAATGTTCCTGTTAC-3’, 5’-CATTCaTCATAGTGAGAATTAGGTGC-3’ that change the codon from GAG to GAT (The changed nucleotides for the overlapping primers are marked as lowercase letters). Primers were made by Evrogen (Moscow, Russia). The changed PCR product was then cloned into the same backbone.\n\nTet-on system was set up to make transcription of the gene of interest to be inducible (Gossen & Bujard, 1992; Gossen et al., 1995). rtTA (tetracycline transactivator), CMV promoter, TRE (Tet Response Element) promoter P2A and RFP were amplified from different plasmids. Influenza A neuraminidase and sialidase from S. typhimurium were inserted under control of TRE-promoter. Neuraminidase was amplified from the plasmid pNAHA (Plasmid # 44169, Addgene) and sialidase S. typhimurium was amplified from pHAGE-CMV-Sal.t.Sia-RFP. All amplified products were cloned in the pHAGE backbone in the following order: TRE-infNA/Sal.t.Sia-P2A-RFP-CMV-rtta by restriction cloning (Supplementary Figure 1B). The plasmid pTagBFP encoding BFP (blue fluorescent protein) was used for cotransfection to approximately estimate effectiveness of transfection before addition of the doxycycline.\n\nCells were treated with Doxycycline (D9891, Sigma; St Louis, MI, USA) at 24 hours after transfection at a concentration of 0.5µg/ml. Expression of the RFP reporter and activity of genes-enzymes were analyzed at 48 hours after Doxycycline addition.\n\nMDCK and HEK293 cell lines were cultured according to ATCC recommendations. Cells were maintained at 37°C with 5% CO2. Cells were transfected with TurboFect reagent (R0531, Thermo Fisher; Waltham, MA, USA) according to the manufacturer recommendations. The fluorescence microscope Axio observer (Zeiss, Oberkochen, Germany) with the standard set of filters was used for visualization of fluorescence. S3e BioRad sorter (Hercules, CA, USA) was used for fluorescence activated cell sorting and flow cytometry. Standard flow cytometry analysis was carried out using FlowJo v10.4 software.\n\nFITC fluorescence in Lectin binding assay was measured by FACS and quantified as an average mean fluorescence intensity (MFI). Values show the means ± SD of triplicate results from representative experiments. Three independent experiments were carried out for each experimental case. Student’s t-test was used to determine the level of statistical significance. Statistical analysis was perform in Microsoft Excel 2016\n\nLentivirus packaging was performed according to the described protocol [see Protocol 1: Lentivirus Packaging by 293T Transfection from CReM].\n\nIn order to determine a viral titre, different aliquots of supernatant were added to HEK293 cells in the presence of 4 μg/ml polybrene (Hexadimethrine bromide, H9268, Sigma). Infection of six 10-fold serial dilutions was performed in 96-well plates. The medium was replaced 12 hours after infection. Three serial infections were performed. At 48 hours after infection, transduction effectiveness was calculated by FACS analysis. The viral titer corresponded to the number of colonies developed at the highest dilution.\n\nThe supernatant of the known titre was used for viral transduction of the MDCK cell line. On the first day about 1.0 x 10-4 MDCK cells were seeded into 6-well plates. The cells were incubated 18–20 hours at 37°C in a humidified incubator in an atmosphere of 5–7% CO2. Upon transduction the confluency around 30–50% was estimated. Next, the appropriate volumes of unconcentrated virus and polybrene with concentration 5ug/ml were added to each well. After 48 hours, cells were analyzed using Flow cytometry to define the percentage of infected/fluorescent cells. Lentiviral transduction efficiency was calculated as (the number of RFP-positive cells/the total number of cells counted × 100) and the number of transduced cells (transduction efficiency (%) × the total number of RFP recovered/100).\n\nGenomic DNA was isolated using Wizard® Genomic DNA Purification Kit (A1120, Promega, Madison, WI, USA) according the manufacturer protocol.\n\nLectin binding assay was used for the cell surface SA detection. For lectin binding assay we used Maackia amurensis (MA) lectins that are specific for α (2–3)-bound SA. The lectin were conjugated with the FITC fluorophore (21761036-1 (510183), bio-WORLD; Dublin, OH, USA). The assay was made for HEK293 and MDCK cells. The experiment was setup in 48-well plates. The cells expressing a desired construct were washed three times with PBS. Then cells were incubated with MA lectins (1:100) about 1 hour and three times washed with PBS. Hoechst (RRID:AB_2651133, Thermo Fisher) was added for visualization of the nuclei for 5 min at recommended concentration with the subsequent washing with PBS.\n\n\nResults\n\nSialidases vary in enzyme kinetic and substrate specificity to the type of the SA linkage. Several sialidases from bacteria, Influenza virus and vertebrate were selected for our study. We were mostly interested in α(2–3) specialized sialidases, because avian Influenza prefers this type of linkage (Ito, 2000). However the broad substrate specificity was also an area of interest. Regarding enzyme kinetics, sialidases with various levels of activity were useful for us in order to combine them with tissue-specific promoters and find an optimal level of desialilation. The optimal sialic acids level on the cells surface prevents penetration of the virus into the cell and does not affect cell function.\n\nSubstrate specificity of bacterial sialidases is very diverse, in terms of type of sialic acid linkage and enzyme kinetics. We selected two different bacterial sialidases: Salmonella typhimurium sialidase, which specializes in cleavage of α(2–3) sialic acid residues (Hoyer et al., 1991; Rogerieux et al., 1993) and Actinomyces viscosus sialidase, which has a substrate specificity to both α (2–3) and α (2–6), but preferentially cleaves the α (2–6) linkage (Teufel et al., 1989). We used sequence of the catalytic domains of the sialidases and added to their N-terminal sequences the transmembrane domain with the stem loop of Influenza NA transmembrane protein in order to target the proteins to the membrane and demonstrate its catalytic activity outside of the cell.\n\nThe human Influenza neuraminidase (sialidase) has been taken for analysis as a broad-spectrum sialidase. Unlike human HA that preferentially recognises α (2–6), the viral NA of H1N1 has cleavage activity to both (2-6) and α(2–3) types of linkages.\n\nExogenous overexpression of a vertebrate membrane sialidase also would be interesting to analyze. The sialidases are poorly investigated, but some experimental data exists for human membrane neuraminidase hNeu3 (Monti et al., 2000; Zhang et al., 2010) and the enzyme was selected for cloning.\n\nAll cloned genetic constructs were firstly tested by transient expression in the HEK293 cell line that has glycosylation patterns including α (2–3) and α (2–6) sialic acid linkages (Picanco-Castro et al., 2013). All plasmids had the RFP reporter to mark the cells that express a sialidase. Using lectin binding assays it has been shown that the cells expressing the sialidase from S. typhimurium have decreased level of SA. Expression of the sialidase from A. viscosus did not affect the α (2–3) sialylation level, probably because it is more specific to the α (2,6) sialic acid linkages. However, it has previously been shown that this sialidase effectively removes both α (2,3)- and α (2,6)-linked SA from the cell surface (Malakhov et al., 2006). Human neuraminidase hNEU3 also has not shown activity towards α (2–3)-linked SA. We suppose that it might be related to unsuitable pH conditions. Cultivation in DMEM in 5% CO2 provides pH in range of 7,6–7,7. Earlier, hNeu3 activity was demonstrated in the pH range of 2.8–6.6 (Monti et al., 2000). In the case of influenza A neuraminidase, sialylation level decreased, not only in RFP-positive cells, but also in RFP-negative cells, which are not supposed to express the enzyme (Figure 2A). According to the flow cytometry analysis, the most significant α (2–3)-linked SA cleavage was in the case of Influenza A neuraminidase expression (Figure 2B). Representative histograms from three independent repeats are shown. Lectin binding assay was quantified as fluorescence and calculated as a mean fluorescence intensity - MFI (±SD): d_infNA-RFP - 103.437 (±3.568), Act.v.Sia-RFP - 181.401(±7.988), Sal.t.Sia-RFP - 7.341(±1.323), infNA-RFP - 4.725(±983), hNeu3 - 27.379(±2.231).\n\nGenetic constructs were transiently expressed in the HEK293 cell line, and sialidase activity was evaluated using a lectin binding assay with FITC-labelled Maackia amurensis lectins. The plasmid with catalytically inactive neuraminidase was used as the negative control. (A) Fluorescent microscopy analysis. Scale bar = 10 um. (B) FACS analysis.\n\nA responsive transcription of a sialidase gene gives an opportunity to induce the gene expression in response to a molecule addition. In our case we used Tetracycline-Controlled Transcriptional Activation where transcription is reversibly turned on in the presence of the antibiotic tetracycline derivative doxycycline. The method can be a useful model for studying viral infection in cell culture and especially for use in genetically modified organisms because constitutive exogenous gene expression could have a potential harm.\n\nFor tetracycline-inducible expression we selected only sialidases that demonstrated significant α (2–3) catalytic activity in the previous experiment with transient expression under CMV promoter (infNA-RFP and Sal.t.Sia-RFP). The plasmid coding BFP under constitutive CMV promoter was used for estimation of transfection effectiveness. It was shown that upon treatment of cells with doxycycline the sialidases expression switched on and as a result the α (2–3) sialylation level decreased (Figure 3A, B). From flow cytometry analysis representative histograms from three repeats are shown. MFI (±SD) was calculated: infNA-RFP, +dox - 1.468 (±439), Sal.t.Sia-RFP, +dox - 6.437(±1.043), d_infNA-RFP, +dox - 83.849(±2.983).\n\nGene expression was evaluated in the HEK293 cell line after doxycycline induction, using a lectin binding assay with FITC-labelled Maackia amurensis lectins. The plasmid with catalytically inactive neuraminidase was used as the negative control. (A) The plasmid coding BFP was used to estimate the effectiveness of transfection. Fluorescent microscopy analysis. Scale bar = 50 um. (B) FACS analysis.\n\nThe MDCK cell line is used for propagation of influenza viruses. To obtain cell lines that stably express exogenous genes coding for different sialidases we transduced MDCK cells with lentiviruses. The expressing construct was integrated into the genome by lentiviral transduction. The integration was confirmed with PCR (Supplementary Figures 2 and 3) using primers from Table 1. The effect of expression of sialidases was studied by testing the reactivity of cells with sialic acid linkage-specific lectins.\n\nThe results confirmed the previously obtained data with the HEK293 cell line. The cells expressing S. typhimyrium sialidase and Influenza A neuraminidase had decreased level of the surface α (2–3) SA. However, the decrease in the SA level in the MDCK was less significant compared with the HEK293 cell line (Figure 4B). Representative histograms from three technical repeats are shown. MFI was calculated: d_infNA-RFP - 38.120(±2.322), Act.v.Sia-RFP - 22.750(±3.175), Sal.t.Sia-RFP - 21.732(±4.598), infNA-RFP - 7.290(±1.934), RFP-NA - 12.798(±3.453). Similarly with the experiments on the HEK293 cell line, in the case of infNA-RFP expression α (2–3) SA were absent not only in the RFP-positive cells but in the RFP-negative cells as well. A possible explanation of the evidence could be a ‘slipstream’ translocation in a polycistronic vector of the P2A downstream protein without a signal of localization that is RFP in our case (de Felipe et al., 2010). An alternatively explanation being when a ribosome encounters 2A within an open reading frame the synthesis of a specific peptide bond could be “skipped”. This results in termination of translation at the end of 2A peptide (Donnelly et al., 2001). It means that RFP is not expressed and can explain the absence of sialic acids in some RFP-negative cells. The effects were not observed in the case of other sialidases. Usage of a longer sequence for P2A (with a favorable upstream sequence composition) or modifying the order of proteins could solve the problem. When we changed the order of the RFP and the infNA sequences (RFP-P2A-infNA), the result of lectin binding assay was as expected: RFP-positive cells showed decreased level of α (2–3) SA (Figure 4A). Nevertheless, in the case of inverted position of the neuraminidase the enzyme activity was less pronounced. It can be explained by the fact that a protein located at the second position can be slightly less expressed in a polycistronic construct with a 2A peptide (Liu et al., 2017). In the case of the S. typhimyrium sialidase expression (Sal.t.Sia-RFP) the ‘slipstream’ effect was not observed.\n\nMDCK cells were transduced with lentiviruses. Lectin binding assay was made with FITC-labelled Maackia amurensis lectins. The plasmid with catalytically inactive neuraminidase was used as the negative control. (A) Fluorescent microscopy analysis. Scale bar = 50 um. (B) FACS analysis.\n\n\nDiscussion\n\nSialic acid is the sole receptor of Influenza A virus (Matrosovich et al., 2013). Therefore the removal of sialic acids from the cell surface might be a powerful defence strategy against Influenza virus. In the current study we take a variety of sialidases from different sources and compared their activity. We demonstrated that expression of the sialidase catalytic domain from Salmonella typhimurium fused with the transmembrane domain and human Influenza A neuraminidase effectively removes α (2–3)-sialic residues on the cell surface under physiological conditions.\n\nIn our study, about 70–80% of the surface SA were removed by the membrane sialidases. It has been reported that the virus binding could still occur with such sialylation level, as has been shown in the MDCK cell line, but amplification of Influenza virus was still inhibited (Stray et al., 2000), therefore complete elimination of SA is perhaps unnecessary. Neuraminidase had higher cleavage activity against α (2–3)-linked sialic acids than sialidase from S. typhimyrium according to our results. An optimal level of desialilation can be established in the tissue when combining genetic constructs with varying sialidase activity and tissue-specific promoters.\n\nStable expression of sialidases potentially could disrupt physiological functions required for proper glycosylation (Gutierrez et al., 1987; Michalek et al., 1988; Pangburn et al., 2000; Varki, 1992). Inducible expression may be more suitable in this case. The Tet-on inducible system allowed robust expression and functioning of genetic constructs encoding sialidases after the inductor addition. The level of sialilation will return to the previous level during a period of time after withdrawal of doxycycline. The required time depends on the sialic acid turnover rate.\n\nIt has been shown that sialidase treatment does not affect the properties of respiratory mucus, nor did it affect the normal mucus transport activity on ciliated epithelium (King et al., 1974; Meyer et al., 1975). Thus, temporary desialilation of the epithelial surface should not cause problems.\n\nOur current preliminary in vitro data indicates that sialidases from Salmonella typhimurium and neuraminidase from Influenza A virus could be the potential candidates that provide antiviral defense against avian Influenza virus. Since sialidases target cellular receptors but not a viral gene product, the chance of influenza viruses developing resistance is low.\n\n\nData availability\n\nData underlying this study is available from Dataverse - doi:10.7910/DVN/UCDPX3 (Antonova, 2018).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work has completed with the financial support of Russian Science Foundation within project No. 16-16-04094.\n\n\nReferences\n\nAir GM, Laver WG: Red cells bound to influenza virus N9 neuraminidase are not released by the N9 neuraminidase activity. Virology. 1995; 211(1): 278–284. PubMed Abstract \| Publisher Full Text\n\nAntonova E: Evaluation of defence strategy against Influenza A in cell line models. Harvard Dataverse, V1 2018. Publisher Full Text\n\nBergelson LD, Bukrinskaya AG, Prokazova NV, et al.: Role of gangliosides in reception of influenza virus. Eur J Biochem. 1982; 128(2–3): 467–474. PubMed Abstract \| Publisher Full Text\n\nde Felipe P, Luke GA, Brown JD, et al.: Inhibition of 2A-mediated ‘cleavage’ of certain artificial polyproteins bearing N-terminal signal sequences. Biotechnol J. 2010; 5(2): 213–223. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDonnelly ML, Luke G, Mehrotra A, et al.: Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'. J Gen Virol. 2001; 82(Pt 5): 1013–25. PubMed Abstract \| Publisher Full Text\n\nGossen M, Bujard H: Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992; 89(12): 5547–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGossen M, Freundlieb S, Bender G, et al.: Transcriptional activation by tetracyclines in mammalian cells. Science. 1995; 268(5218): 1766–9. PubMed Abstract \| Publisher Full Text\n\nGriffin JA, Basak S, Compans RW: Effects of hexose starvation and the role of sialic acid in influenza virus release. Virology. 1983; 125(2): 324–334. PubMed Abstract \| Publisher Full Text\n\nGutierrez C, Martin MJ, Brown KA: Complement activation by human lymphocytes from different lymphoid organs: role of sialic acid and lack of relationship to electrical surface charge. Complement. 1987; 4(2): 99–109. PubMed Abstract \| Publisher Full Text\n\nHoyer LL, Roggentin P, Schauer R, et al.: Purification and properties of cloned Salmonella typhimurium LT2 sialidase with virus-typical kinetic preference for sialyl alpha 2----3 linkages. J Biochem. 1991; 110(3): 462–7. PubMed Abstract \| Publisher Full Text\n\nHuang IC, Li W, Sui J, et al.: Influenza A virus neuraminidase limits viral superinfection. J Virol. 2008; 82(10): 4834–4843. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIto T: Interspecies transmission and receptor recognition of influenza A viruses. Microbiol Immunol. 2000; 44(6): 423–30. PubMed Abstract \| Publisher Full Text\n\nKim JK, Negovetich NJ, Forrest HL, et al.: Ducks: The “Trojan Horses” of H5N1 influenza. Influenza Other Respir Viruses. 2009; 3(4): 121–128. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKing M, Gilboa A, Meyer FA, et al.: On the transport of mucus and its rheologic simulants in ciliated systems. Am Rev Respir Dis. 1974; 110(6): 740–5. PubMed Abstract\n\nLiu Z, Chen O, Wall JBJ, et al.: Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep. 2017; 7(1): 2193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatrosovich M, Herrler G, Klenk HD: Sialic Acid Receptors of Viruses. Top Curr Chem. 2013; 367: 1–28. PubMed Abstract \| Publisher Full Text\n\nMatrosovich MN, Matrosovich TY, Gray T, et al.: Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci U S A. 2004; 101(13): 4620–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalakhov MP, Aschenbrenner LM, Smee DF, et al.: Sialidase Fusion Protein as a Novel Broad-Spectrum Inhibitor of Influenza Virus Infection. Antimicrob Agents Chemother. 2006; 50(4): 1470–1479. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeyer FA, King M, Gelman RA: On the role of sialic acid in the rheological properties of mucus. Biochim Biophys Acta. 1975; 392(2): 223–32. PubMed Abstract \| Publisher Full Text\n\nMichalek MT, Bremer EG, Mold C: Effect of gangliosides on activation of the alternative pathway of human complement. J Immunol. 1988; 140(5): 1581–7. PubMed Abstract\n\nMonti E, Bassi MT, Papini N, et al.: Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane. Biochem J. 2000; 349(Pt 1): 343–351. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoscona A: Neuraminidase inhibitors for influenza. N Engl J Med. 2005; 353(13): 1363–73. PubMed Abstract \| Publisher Full Text\n\nPalese P, Tobita K, Ueda M, et al.: Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology. 1974; 61(2): 397–410. PubMed Abstract \| Publisher Full Text\n\nPangburn MK, Pangburn KL, Koistinen V, et al.: Molecular mechanisms of target recognition in an innate immune system: interactions among factor H, C3b, and target in the alternative pathway of human complement. J Immunol. 2000; 164(9): 4742–51. PubMed Abstract \| Publisher Full Text\n\nPicanco-Castro V, Biaggio RT, Cova DT, et al.: Production of recombinant therapeutic proteins in human cells: current achievements and future perspectives. Protein Pept Lett. 2013; 20(12): 1373–81. PubMed Abstract \| Publisher Full Text\n\nRogerieux F, Belaise M, Terzidis-Trabelsi H, et al.: Determination of the sialic acid linkage specificity of sialidases using lectins in a solid phase assay. Anal Biochem. 1993; 211(2): 200–4. PubMed Abstract \| Publisher Full Text\n\nSkehel JJ, Wiley DC: Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem. 2000; 69: 531–69. PubMed Abstract \| Publisher Full Text\n\nStray SJ, Cummings RD, Air GM: Influenza virus infection of desialylated cells. Glycobiology. 2000; 10(7): 649–58. PubMed Abstract \| Publisher Full Text\n\nTeufel M, Roggentin P, Schauer R: Properties of sialidase isolated from Actinomyces viscosus DSM 43798. Biol Chem Hoppe Seyler. 1989; 370(5): 435–43. PubMed Abstract \| Publisher Full Text\n\nVarghese JN: Development of neuraminidase inhibitors as anti-influenza virus drugs. Drug Dev Res. 1999; 46(3–4): 176–196. Publisher Full Text\n\nVarki A: Selectins and other mammalian sialic acid-binding lectins. Curr Opin Cell Biol. 1992; 4(2): 257–66. PubMed Abstract \| Publisher Full Text\n\nVarki A, Cummings RD, Esko JD, et al.: Essentials of glycobiology, 2nd edn. Cold Spring Harbor Laboratory Press, New York. 2009. PubMed Abstract\n\nZhang M, Koskie K, Ross JS, et al.: Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells. Biotechnol Bioeng. 2010; 105(6): 1094–1105. PubMed Abstract \| Publisher Full Text" }	[ { "id": "31110", "date": "12 Mar 2018", "name": "Gillian M Air", "expertise": [ "Reviewer Expertise Influenza" ], "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 expressed membrane-anchored constructs of influenza, human and bacterial sialidases in cells and measured the effect on cell surface sialylated glycans. The study is preliminary but well executed as far as it goes.\n\nGeneral comments:\nThe manuscript does not include evaluation of influenza virus infection so the title is exaggerated. Maybe “Evaluation of potential defense strategy…”. would be less misleading. The Figure legends are very terse and a reader needs to make some guesses at what is being shown. For example, what are the boxes on the panels in Figures 3 and 4? The manuscript needs to be checked for spelling. “sialilation” appears several times and there are other errors such as S. typhimyrium on p.7.\n\nSpecific comments:\nAbstract: I have not seen data that shows the H5N1 virus is increasingly infective for humans. Either reference this statement or modify it. Abstract and text: the increased activity seen with influenza neuraminidase is likely due to the construct being more native and better folded. The S. typhimurium sialidase itself has much higher activity than influenza NA. P.5: the Maakia amurensis lectins have been variable between suppliers and one form binds additional glycans, as has been discussed in several recent publications. More information is needed to be sure the lectin used has alpha2-3 specificity. P.5, first paragraph of Results. “The optimal sialic acids level on the cells surface”. I think this should refer to sialidase, not sialic acid. P.5 The influenza NA does indeed cleave alpha2-6 linked sialic acid but at 1/3 to 1/5 the rate of alpha2-3. P.5: while the assay to show reduction of cell surface sialic acids fits the goal of the project, it might be useful to carry out classical enzyme assays using the expressed sialidases to determine if the constructs used have similar activity to their wild type counterparts and if not, if they can be improved.\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?\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": "3631", "date": "16 May 2018", "name": "Ekaterina Antonova", "role": "Author Response", "response": "Respond to the general comments: 1.The correction has been done in the version 2. 2.Figure legends have been supplemented with details in the version 2. 3. The manuscript has been checked for spelling and found errors and typos were corrected in the version 2. Respond to the specific comments: 1.The statement was modified to ‘Avian H5N1 and H7N9 virus has a potential pandemic threat’ in the version 2. 2.The information was added in the Results/Transient expression of genetic constructs in HEK293 cell lines. 3.Maakia amurensis lectins from bioWORLD that have declared alpha(2;3) specificity were used as analogous to an avian hemagglutinin due to they share similar structural fold. The lectins were used for preliminary study and it is planned to continue the research with an avian virus. 4. Both statements make sense, however “the optimal sialic acids level” is the result of sialidases expression and it undermines not only the level of sialidases on the cell surface but their catalytic activity. 5.The statement «Unlike human HA that preferentially recognises α (2–6), the viral NA of H1N1 has cleavage activity to both (2-6) and α(2–3) types of linkages» was supplemented with the information that NA also cleaves alpha2-6 linked sialic acids at lower rate than alpha2-3 . 6.The aim of the project was to compare created genetic constructs between each other and use some of them in future researches. We didn’t try to achieve similar activity of the constructs to their recombinant counterparts. However it would be interesting to check how folding and localisation influence on the enzymes activity." } ] }, { "id": "32149", "date": "05 Apr 2018", "name": "Laurence S. Tiley", "expertise": [ "Reviewer Expertise Influenza molecular virology. Transgenic livestock." ], "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\nSummary: This paper reports the construction of some vectors to express various sialidases with the ultimate intention of generating transgenic chickens expressing these genes.\n\nThe strategy is intended to protect chickens against influenza virus by eliminating the receptor for the virus. The vectors were tested transiently in 293T cells and then in stable MDCK cell lines. Expression of the influenza NA and S.typhimurium NA reduced alpha 2-3 sialic acid levels at the cell surface as judged by lectin staining.\nI see very little of publishable merit at this stage of the work and the quality of the text requires substantial improvement.\nSpecific comments: Abstract: 1. H5N1 is not showing increasing infection/adaptation for humans. 2. I suggest the authors consider the increased threat posed by H7N9 which is arguably of far more concern at present.\nIntroduction: 3. The cumulative case count for H5N1 is 454 deaths out of 860 total cases. It is unlikely that the case count of a disease with this high case fatality rate has caused \"several thousand\" cases and \"some with fatal outcome\". The wording here is very imprecise. 4. The citation is from 2005 and totally inappropriate. See: http://www.who.int/influenza/human_animal_interface/2018_03_02_tableH5N1.pdf?ua=1\n5. Seasonal flu vaccination is primarily used to protect individuals in high risk groups. Its impact on spread in the general population is probably insignificant.\nMethods: 6. The construction details for RFP-P2A-infNA are not provided. 7. The method for the determination of the lentivector titre is unclear. The analysis by FACS is OK, but what are the authors referring to when they mention \"The viral titre corresponded to the number of colonies developed at the highest dilution?\" 8. What exactly do they mean by \"three serial infections\"? 9. Typo: they used ~1x104, not 1x10-4 MDCK cells.\nNote: I could not access all the supplementary info (even on the Harvard Dataverse).\nResults: 10. d_infNA-RFP is not defined, but presumably it is the inactive E262D. 11. Surely a typo for the SD value here: infNA-RFP - 4.725(±983) 12. Before making more complex speculations regarding the inactivity of the hNeu3 and Act.v.Sia constructs, the authors should demonstrate they are expressed in the correct compartment. RFP expression merely shows the mRNA were translated.\n\n13. Another typo for the SD: infNA- RFP, +dox - 1.468 (±439)?\n14. The consistent apparent activity of the N1 neuraminidase acting on non-transduced cells, could more simply be an indication that the protein is being secreted. The lack of data on protein localisation does not help with the interpretation. The \"slip-stream\" explanation hypothesising that many more cells are transduced than were apparent in the photos is possible, but not very credible as this would also need the lentivirus titre/transduction efficiency to be much higher for the infNA vector. 15. Little consideration regarding the topology of their constructs has been given.\nDiscussion:\n16. The discussion is quite superficial. The authors might like to mention related approaches such as DAS181 and the discussions regarding this general approach1\n17. There are reports that de-sialylation does not completely abrogate influenza infectivity. A point perhaps worth mentioning briefly.\n18. They may wish to mention more about the possible physiological consequences e.g. the potential impact on the MCE of constitutively expressing a sialidase2\n19. The choice of appropriate promoter is not discussed in any detail. Presumably the intention is to restrict expression to relevant tissues i.e. the respiratory and GI tract in the case of poultry. Do they have a particular promoter in mind?\n\n20. The practicalities of using a Dox-inducible promoter are not considered. Is the intention to treat flocks continuously with Dox to prevent infection, or to respond prophylactically to an outbreak in a nearby location? The use of Dox in this way is undesirable for several reasons.\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? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "3634", "date": "16 May 2018", "name": "Ekaterina Antonova", "role": "Author Response", "response": "Abstract 1-2. Agreed. The statement regarding H5N1 was modified to ‘Avian H5N1 and recently H7N9 virus has a potential pandemic threat.’ in the version 2. Introduction 3.The sentence has been corrected from “Several thousands of cases of human infection were recorded and many of them with a fatal outcome” to “860 cases of disease in human population have been detected since 2003 and 454 of them with fatal outcome (see http://www.who.int/influenza/human_animal_interface/2018_03_02_tableH5N1.pdf?ua=1 4. The reference has been changed in the version 2. 5. Agreed. The sentence has been corrected Methods 6. The information has been added in the Methods part: “Additionally the reverse construction RFP-P2A-infNA was cloned in the same vector. (Supplementary Figure 1A)”. 7. The sentence \"The viral titre corresponded to the number of colonies developed at the highest dilution\" was deleted due to it is related to the titre determination with drug selection. However lentiviral constructions with resistant genes were not considered in the publication. Instead, the following text has been added in the version 2: “FACS analysis was performed using non-infected cells as a negative control. The cells with 10%-40% RFP-positive were selected for titer calculation. The titer was calculated using the equation: Titer (Transduction Unit /ml) = (Cell Number in each well used for infection on Day 2 × percentage of GFP/RFP positive cells (should be 10%-40%))/(virus volume used for infection in each well × dilution fold)” 8. The three repeats were mentioned. The phrase \"three serial infections” was corrected in the text to avoid confusion: “Infection with the same aliquot was used in three repeats.” 9. The typo has been corrected in the version 2. Please check the supplementary availability now. Supplementary Figure 1A & 1B. Genetic constructs. Click here to access the data. Supplementary Figure 2. Confirmation of inserted genes in MDCK genome after lentiviral transduction. Click here to access the data. Supplementary Figure 3. Schematic of integrated constructs with primers for PCR analysis. Click here to access the data. Results 10.The mention has been added in the part Methods/Catalytically inactive sialidaseю 11. Corrected: 4.725(±0.983) 12. We used well-known transmembrane domain of Influenza neuraminidase for proper localization of both Act.v.Sia and Sal.t.Sia sialidases on cytoplasmic membrane. The sequence of the transmembrane domain and stem loop was the same for both bacterial sialidases, however only one was active. It is known that the sialidase hNeu3 is localised on the plasma membrane. We cloned the gene and checked the sequence. Lectin binding assay was used to confirm its functionality on the cell surface hence the correct localisation (in the case if activity was demonstrated). However we agree with your point and we have noticed it in the text (Results/Transient expression of genetic constructs in HEK293 cell lines) 13. Corrected: 1.468 (±0.439) 14. We have considered the hypothesis regarding secreted neuraminidase and rejected it. First, because we used neuraminidase from Influenza Puerto Rico strain (Addgene). And it is well-known that the neuraminidase is located on the plasma membrane surface. We sequenced the neuraminidase gene after cloning. Second, the reversion of positions of neuraminidase and RFP would not influenced on neuraminidase secretion. But in our case only RFP-positive cells were desialylated (CMV-RFP-P2A-infNA expression) that exclude secretion. Regarding the lentivirus titre/transduction efficiency. The lentivirus titre was defined by RFP fluorescence. However not all RFP sequences could be translated properly due to the effect of termination of translation at the end of 2A peptide that resulted in lower RFP fluorescence. Hence the lentivirus titre could be underestimated. In result the cells were infected by larger quantity of viral particles. FACS data was normalised to RFP fluorescence level when analysing the level of lectin binding. 15. We used well-characterised transmembrane domain to target all the enzymes (except human Neu3) on the cell surface and made functional estimation of catalytic activity using lectin binding assay. Enzymatic activity of a protein in the cell expressing reporter confirms membrane location of the protein on the cell membrane. Discussion 16. The mention regarding DAS181 has been added in the discussion part: “Previously, a recombinant sialidase (DAS181) has already demonstrated its antiviral effectivity in the cell culture (Malakhov MP et al., 2006)) and in vivo, using a mouse model (intranasal injection of the recombinant fusion protein - DAS181) (Belser JA et al., 2007). Thus, the principal possibility of the usage of membrane-anchored sialidase as antiviral defence has been demonstrated.” 17. We added in the discussion points regarding the possible risk: \"There are several concerns to use the strategy. The first is that influenza virus receptors may be different from SA, thus, sialidase expression may be ineffective. It has been reported that the virus binding could still occur in the MDCK cell line desialylated with addition in the medium Micromonospora viridifaciens sialidase ( Stray et al., 2000). The authors suggested that influenza virus infection can result from sialic acid–independent receptors, either directly or in a multistage process. The presence of sialic acid may enhance virus binding to the cell surface to increase interaction with secondary receptors to mediate entry. Also the authors mentioned that high multiplicity of infection has an increased requirement for sialic acids and desialylation will inhibit the virus amplification. Although the HA of a human H1N1 strain was shown to bind other glycoconjugates (Rapoport et al., 2006), the exact receptors to enter into the cell have not been found. Stray et al., used NA-deficient virus in the research which was obtained in a laboratory. It is unlikely that such virus would exist in nature. Probably, the barrier to efficient infection and transmission between organisms is due to inefficient use or expression of a yet unidentified entry mediator.\" 18. Added: “Another concern of the stable expression of sialidases is disruption of physiological functions required for proper glycosylation ( Gutierrez et al., 1987; Michalek et al., 1988; Pangburn et al., 2000; Varki, 1992). The probable consequences of constitutive expression of membrane sialidase in vivo are not known. It has been shown that overexpression of the human ortholog NEU3 membrane sialidase under the β-actin promoter in transgenic mice resulted insulin-resistant diabetes mellitus (Sasaki et al., 2003). The authors focused on insulin signaling only. However at least the data shows that the constitutive membrane sialidase expression was not lethal for mice. We are going to reduce a probable negative impact on organism using tissue-specific expression of the selected gene. It is necessary to predict a potential impact on the epithelium of intestine and trachea in vivo. There are no similar genetic experiments. However the consequences of tissue-specific expression could be considered from the researches where local desialylation was done. It has been shown that intranasal sialidase treatment does not affect the properties of respiratory mucus, nor did it affect the normal mucus transport activity on ciliated epithelium (King et al., 1974; Meyer et al., 1975). Some authors showed that Influenza infection is associated with an increased risk of secondary infection by Streptococcus pneumoniae (Sluijs et al., 2006). There are concerns that, probably, tissue-specific expression of sialidases would result in a greater chance of bacterial infection in the respiratory tract due to exposure of its binding sites that were masked by terminal sialic acid. It has been shown that the binding sites for the bacteria already exist on the normal respiratory surface and sialidase treatment does not introduce de novo bacterial adhesion sites. Also the risk of infection arises due to damage of mucus and epithelial cells. But it is not clear whether the secondary S. pneumoniae infection happens due to viral NA activity or from a secondary effects of influenza virus intracellular amplification. Influenza infection in the respiratory tract causes much deeper changes to the epithelium than treatment of the surface of the epithelial cells with a sialidase, because Influenza infection in humans and in mice results in vast exposure of the ciliated epithelial cells down to the basal cells and the basement membrane (Walsh et al , 1961; Plotkowski et al.,1986). Thus, the most likely the increased S. pneumoniae infection is the result of airway epithelial damage caused by the virus infection but not just desialylation. In the gastrointestinal tract SA is an abundant sugar residue in mucin that is a key target of intestinal bacteria. Expression of sailidases will release free SA from mucins that could drives intestinal inflammation and infection. For example, elevated levels of free sialic acid in the gut, during and post antibiotic treatments, promote the expansion of Clostridium difficile, that utilises free SA. Also it has been shown that sialidase activity can promote the outgrowth of Escherichia coli and result in imbalanced microbiota, inflammation, colitis development due to α (2–3)-sialic residues promote expansion of E. coli (Huang et al., 2015). Nevertheless, we do not know the probable negative impact on the physiology of avian respiratory and digestive tract. Thus, an optimal level of desialylation can be established in the tissue when combining genetic constructs with varying sialidase activity and tissue-specific promoters to avoid disruption of physiological functions in our approach. Additionally, a gene expression with time limit could prevent a probable negative impact on a living organism. Michael P. Malakhov et al. showed that temporary desialylation of respiratory tract during 7 days using recombinant sialidase protein did not cause any symptoms of toxicity or inflammation in ferrets and mice that were not infected by the virus (Malakhov et al., 2006). This in vivo data support our idea that temporary inducible expression of a sialidase will not have serious negative physiological effects for transgenic animals.” 19. Yes, we have. We have analysed several promoters specific for intestine and trachea. We have measured expression in scraping of the epithelium from the chicken intestine and trachea by RT-PCR and selected several tissue specific genes that have different expression level. Respiratory tract: SFTPA2, SLC34A2. Related expression normalized to GAPDH: SFTPA2 (3,5±0,7); SLC34A2 (0,002). p<0,001 Digestive tract: RBP2, GUCA2A, MUC13, OLFM4. Related expression normalized to GAPDH: RBP2 (1,1±0,01); GUCA2A (0,4±0,005); MUC13 (0,18±0,05); OLFM4 (0,09±0,015), p<0,01. 20. It is planned to use Dox prophylactically to an outbreak. The practicalities of using Tet-on system were added: “We are planning to use the Tet-on system as a potential model for creation of genetically modified organism that allows to induce a sialidase expression temporary. Transgenic transcription factor rtTA will be expressed under control of a tissue-specific promoter and a sialidase will be expressed under control of TRE promoter. This enables tissue-specific inducible control of the transgene expression and will provide a powerful and flexible resource for studies of influence of sialylation on chicken physiology and on viral infection. The level of sialylation will return to the previous level during a period of time after withdrawal of doxycycline. The required time depends on the sialic acid turnover rate. The inducing agent can be treated prophylactically to genetically modified birds in an outbreak that can protect livestock and locally stop infection spread. It is known that the inducible system has some limitations. Firstly, it has basal transgene ‘leak’. However in our case at least the leak will be in the specific tissue. The probable consequences of the leak will be studied. Secondly, the feeding of domestic birds with the doxycycline antibiotic can compromise its safety as biological products. However it is a temporary measure that could help to save the stock. The efficacy of our strategy needs to be evaluated in vivo to resolve all the concerns.'" } ] } ]	1	https://f1000research.com/articles/7-206
https://f1000research.com/articles/7-599/v1	16 May 18	{ "type": "Software Tool Article", "title": "freesurfer: Connecting the Freesurfer software with R", "authors": [ "John Muschelli", "Elizabeth Sweeney", "Ciprian M. Crainiceanu", "Elizabeth Sweeney", "Ciprian M. Crainiceanu" ], "abstract": "We present the package freesurfer, a set of R functions that interface with Freesurfer, a commonly-used open-source software package for processing and analyzing structural neuroimaging data, specifically T1-weighted images. The freesurfer package performs operations on nifti image objects in R using command-line functions from Freesurfer, and returns R objects back to the user. freesurfer allows users to process neuroanatomical images and provides functionality to convert and read the output of the Freesurfer pipelines more easily, including brain images, brain surfaces, and Freesurfer output tables.", "keywords": [ "freesurfer", "r", "neuroconductor", "neuroimaging" ], "content": "Introduction\n\nFreesurfer is a commonly-used software for processing and analyzing anatomical neuroimaging data1, developed by the Laboratory for Computational Neuroimaging at the Athinoula A. Martinos Center for Biomedical Imaging. This software provides open-source, command-line tools for image processing tasks such as brain extraction/skull-stripping2, bias-field correction3, segmentation of structures within the brain4,5, and image registration6,7. In addition to these functions, Freesurfer has functions that perform fully-automated pipelines for the user.\n\nThere exist a number of R packages for reading and manipulating image data, including AnalyzeFMRI8 and fmri9, which analyze functional magnetic resonance images (MRI) and perform spatial smoothing, RNiftyReg10, which performs image registration, and dpmixsim11 and mritc12, which perform image clustering and segmentation (see the Medical Imaging CRAN task view for more information). These packages provide powerful tools for performing image analysis, but the neuroimaging community has additional tools that may perform better on a specific data set or provide more information than these R packages. Freesurfer provides methods that are not currently implemented in R, including surface-based registration and completely automated image segmentation pipelines. The ANTsR package is a currently unpublished R package where additional image analysis functionality has been implemented, but does not include all the functionality Freesurfer has. Moreover, having multiple options for image processing through R enables users to compare methods and provides the flexibility of using multiple packages to achieve a working data processing pipeline.\n\nWe provide an interface to users for the state-of-the-art anatomical processing implemented in Freesurfer, as well as a suite of tools that simplify analyzing the output of Freesurfer. The freesurfer package allows R users to perform a complete anatomical imaging analyses without necessarily learning Freesurfer-specific syntax, while keeping both the image processing and analysis within R.\n\n\nMethods\n\nTo use freesurfer, a working installation of Freesurfer is required (downloads available: http://freesurfer.net/fswiki/DownloadAndInstall). The following code was run using Freesurfer version “freesurfer-Darwin-lion-stable-pub-v5.3.0”. The Freesurfer version can be accessed using the freesurfer fs_version function. The path of Freesurfer must also be set. When using R from a shell environment, after the FREESURFER_HOME environment variable is set (which is done when installing Freesurfer), freesurfer will use this as the path to Freesurfer. If using R through a graphical user interface (GUI) such as RStudio (RStudio, Boston, MA), environmental variables and paths are not explicitly exported. Therefore, FREESURFER_HOME is not set and freesurfer will try the default directories of Mac OSX and Linux. Freesurfer is only available on Windows via a virtual machine. If the user did not perform a standard installation of Freesurfer, the path to Freesurfer can be specified using options(freesurfer.path=\"/path/to/freesurfer\"). The have_fs function tests whether a user has a Freesurfer installation, returning a logical, which is useful for if statements within examples. If have_fs function returns is TRUE, the fs_dir function will return the directory of the Freesurfer installation.\n\nAs per the https://surfer.nmr.mgh.harvard.edu/fswiki/DownloadAndInstall, Freesurfer only works on Linux or Mac operating systems. The work station should have at least a 2GHz processor, over 8GB of RAM, over 10GB hard drive space, and FSL13 installed for certain functions.\n\nDuring the installation of Freesurfer, environment variables in addition to FREESURFER_HOME are set. One of these variables is SUBJECTS_DIR, which refers to a directory of the output of analysis from all subjects. The fs_subj_dir function will return the path to the Freesurfer subjects directory if it is set. This default setup of a subjects directory in Freesurfer allows users to simply specify a subject identifier to analyze, rather than a specific path or multiple intermediate files.\n\nThis setup may not be desirable if the user prefers to structure his or her data differently. For example, if data from multiple studies are present, these may be organized into different folders in different locations. Some functions in Freesurfer rely on the SUBJECTS_DIR variable to run. These functions take the subject name as the main argument rather than a file, which is more common. To provide flexibility to the user, freesurfer allows most functions to specify a file or different directory rather than specifying the subject.\n\nOne example is the asegstats2table Freesurfer function. Freesurfer performs segmentations of the anatomical image into different structures and has associated statistics for each region such as volume and mean intensity. The asegstats2table function transforms anatomical segmentation statistics from images into to a table. The default argument for asegstats2table is to pass in a subject name rather than a file. The freesurfer asegstats2table function allows the R user to specify the subject name, but also allows the user to alternatively specify a file name instead. This function will temporarily set SUBJECTS_DIR to a temporary directory, copy the file to that directory, execute the command, then reset the SUBJECTS_DIR variable. This provides a more flexible workflow, while not overriding the default directory set in SUBJECTS_DIR. This functionality allows users to have separate folders with subjects and read in the data by simply switching the subj_dir argument in the R function.\n\nThe Freesurfer pipeline and analysis workflow for neuroanatomical images is designed to work with T1-weighted structural MRI of the brain. The full pipeline is implemented in the Freesurfer recon-all function, where the “recon” stands for reconstruction (https://surfer.nmr.mgh.harvard.edu/fswiki/recon-all). The recon-all function is the main workhorse of Freesurfer and is the most commonly used. Using the -all flag in the the recon-all function performs over 30 different steps and takes 20–40 hours to fully process a subject (https://surfer.nmr.mgh.harvard.edu/fswiki/recon-all). This process is the recommended way of fully processing a T1-weighted image in Freesurfer, and is implemented in the recon_all freesurfer function.\n\nIn the recon_all function, users must specify the input file (a T1-weighted image), the output directory (if different than SUBJECTS_DIR), and the subject identifier. The results will be written in the individual subject directory, a sub-directory of SUBJECTS_DIR. The syntax is:\n\n\n\nIf there are problems with the result of this processing, there are multiple steps where users can edit certain parts of the processing, such as brain extraction, where non-brain tissues are removed from the image. The remainder of the pipeline can be run after these steps are corrected. The full pipeline is broken down into 3 separate sets of steps, referred to as autorecon1, autorecon2, and autorecon3, which correspond to the same-named flags in recon-all used to initiate these steps. We have written wrapper functions autorecon1, autorecon2, and autorecon3, respectively, so users can run pieces of the pipeline if desired or restart a failed process after correction to the data.\n\nThe freesurfer package relies on the oro.nifti14 package implementation of images (referred to as nifti objects) that are in the Neuroimaging Informatics Technology Initiative (NIfTI) format. For Freesurfer functions that require an image, the R freesurfer functions that call those Freesurfer functions will take in a file name or a nifti object. The R code will convert the nifti to the corresponding input required for Freesurfer. From the user’s perspective, the input/output process is all within R, with one object format (nifti). The advantage of this approach is that the user can read in an image, do manipulations of the nifti object using standard syntax for arrays, and pass this object into the freesurfer R function. Thus, users can use R functionality to manipulate objects while seamlessly passing these object to Freesurfer through freesurfer.\n\nOther Freesurfer functions require imaging formats other than NIfTI, such as the Medical Imaging NetCDF (MINC) format. The Freesurfer installation provides functions to convert from MINC to NIfTI formats and these are implemented in functions such as nii2mnc and mnc2nii in R. Moreover, the mri_convert Freesurfer function has been interfaced in freesurfer (same function name), which allows for a more general conversion tool of imaging types for R users than currently implemented in native R. Thus, many formats can be converted to NIfTI and then read into R using the readNIfTI function from oro.nifti.\n\n\nExample analyses and use of functions\n\nFor this paper, we will not run the analysis on a subject, but rather explore the output results for a subject included in the Freesurfer installation for reproducibility for the user. In particular, in the default Freesurfer subjects directory, there is a subject named “bert”. If we were to run all the analyses, we would use the recon_all code (described below):\n\n\n\nWe see the result of this output in the “bert” directory, which includes a series of sub-directories:\n\n\n\nWe will explore the results in “mri”, which contain imaging data, “stats”, which containing statistics based on structures of the brain, and “surf”, which contain the surface and curvature output from the Freesurfer processing.\n\nThe typical output format of brain volumes from Freesurfer is MGH/MGZ format, which is explained here: https://surfer.nmr.mgh.harvard.edu/fswiki/FsTutorial/MghFormat. As NIfTI formats are one of the most common formats and has been the common format for analysis in the oro.nifti and neurobase packages, it is useful to convert these files to a NIfTI format to read into R. The mri_convert Freesurfer function will be used for that conversion. Here we will use the T1-weighted image from the “bert” subject and convert it to NIfTI, and read it into R:\n\n\n\nAs this is a commonly-used process, we have wrapped these two steps into the readmgz and readmgh functions, which combine the mri_convert and readnii functions. Here we show that these steps are equivalent to the readmgz function:\n\n\n\nNow that we have the image in R, we can plot it using the standard plotting tools for nifti objects:\n\n\n\nThe result is in Figure 1, which contains 3 slices of the head: axially, viewing the brain from the top of the head (top left), sagittally, viewing the brain from the right side (top right) and coronally, viewing the brain from the back of the head (bottom left).\n\nNote, the image is not stored in the right/posterior/inferior (RPI) orientation which is assumed when displaying using the neurobase ortho2 function. We can use the rpi_orient function in fslr (version ≥ 2.4.0)15 or fslswapdim to reorient the image to the assumed orientation.\n\n\n\nWe see that this function puts this image in the RPI orientation, which matches the assumed orientation for ortho2:\n\n\n\nThe result is in Figure 2, which changes the views in reference to which panel they are located and matches the orientation markers assumed by ortho2.\n\nNote, the letters denote the orientation of right/left (R/L), posterior/anterior (P/A), inferior/superior (I/S).\n\nMRI images typically exhibit good contrast between soft tissue classes, but intensity inhomogeneities in the radio frequency field can cause differences in the ranges of tissue types at different spatial locations (e.g. top versus bottom of the brain). These inhomogeneities/non-uniformities can cause problems with algorithms based on histograms, quantiles, or raw intensities16. Therefore, correction for image inhomogeneities is a crucial step in many analyses. The Freesurfer function nu_correct performs the non-uniformity correction by Sled et al.3, and the freesurfer function of the same name will run the correction and return an image. The Freesurfer nu_correct function requires a MINC format (http://www.bic.mni.mcgill.ca/ServicesSoftware/MINC). For this to work, you can convert the nifti object to a MINC file using nii2mnc:\n\n\n\nWe can pass this MINC file into the freesurfer nu_correct function, which will run the correction and then convert the output MNC to a NIfTI object.\n\n\n\nWe see that the results are indeed nifti objects. We can plot the estimated bias field (log-transformed for display purposes) side-by-side with the image to view which areas had been differentially corrected (Figure 3).\n\nIn addition to the readmgz and readmgh functions above, we have a readmnc wrapper function for reading in MINC files, after conversion to NIfTI files. If you pass in a nifti object in directly into nu_correct, the function will automatically convert any NIfTI input files, and then run the correction (shown below). We can also pass in a mask of the brain (see next section) to run the correction only the areas of the brain.\n\n\n\nOverall, this correction is a way to make the intensities of the brain more homogeneous spatially. This method is different from that implemented in FSL13 (and therefore fslr), so it provides an alternative method to the R user than currently available.\n\nThe mri_watershed function will segment the brain from the remainder of the image, such as extra-cranial tissues. Other imaging software in R have implemented the watershed algorithm, such as EBImage17. These methods have not been directly adapted for MRI nor specifically for brain extraction. In freesurfer, we can pass in the nifti object and the output is a brain-extracted nifti object.\n\n\n\nThe result is in Figure 4, where we see areas of the skull, eyes, face, and other areas of the image are removed. We do see some areas remain that may be part of some of the membranes between the brain and the skull, but this looks like an adequate brain extraction for most analyses.\n\nWe see that the areas outside of the brain have been removed from the image.\n\nAs the result is a nifti object, we can create a mask by standard logical operations for arrays. As MRI scans are typically positive-valued, the positive areas of the image are the “brain”:\n\n\n\nWe can then use this mask to perform operations on the image, such as subsetting.\n\nFreesurfer is commonly used to segment cortical and subcortical structures of the brain. We can visualize images of these segmentations, which are located in the “mri” folder. We will choose the colors based on the Freesurfer look up table (LUT), which values can be explored at https://surfer.nmr.mgh.harvard.edu/fswiki/FsTutorial/AnatomicalROI/FreeSurferColorLUT. This look up table provides a label for each structure and the color associated with it:\n\n\n\nThis object is included in freesurfer and denotes the indices, labels, and color representation of the structure. We note that the alpha channel is set to 0 for all regions of interest, so we will not use it in the calculation of the colors from RGB space. This LUT allows visualizations produced in R to be consistent with those from Freesurfer.\n\n\n\nNote above that the number of breaks must be one larger than the number of colors and the indices start at zero, so we add an additional element to the indices. The result in Figure 5 shows the image with colors overlaid.\n\nWe have explored the spatial results in the brain images, but not the quantitative information about the brain and sub-structures that are available from Freesurfer output. The “aseg.stats” in the “stats” folder for subject bert corresponds to measures and statistics from the anatomical segmentation. The read_aseg_stats function reads this corresponding file and creates a list of two different data.frames:\n\n\n\nThe measures element corresponds to global measurements of the brain (e.g. volume of the brain) as well as measures of gross anatomical structures (e.g. gray matter).\n\n\n\nIn some imaging analyses, comparing at these large measures of brain volume over time or across groups are of interest. Alternatively, the structures element corresponds to a set of measures and statistics for a set of fixed anatomical structures.\n\n\n\nSimilarly with global measures, these structure-specific measures are used in analysis. For example, measuring differences in hippocampus volumes across patients with Alzheimer’s disease and those without. Moreover, a large deviation in volume, globally or locally, for a specific subject may indicate atrophy of a structure or an indication of a segmentation error.\n\nFreesurfer includes segmentations of different surfaces of the brain alongside the volumetric segmentations above. As the mri_convert function provides a tool to convert image volumes to a series of output formats, the mris_convert (note the “s”) allows users to convert between image surface formats. These surfaces usually store sets of vertices and faces to be plotted in 3 dimensions. freesurfer has implemented mris_convert (with a function of the same name) as well as functions to convert surfaces from Freesurfer to a set of triangles in R, such as surface_to_triangles. We will read in the left and right side of the pial surface of the brain and display the surface using rgl18 (Figure 6).\n\n\n\nThus, we can read in the output images, surfaces, and the tables of output metrics from Freesurfer.\n\nFor the initial release, we did not implement a method to read the annotation files and other surface-based files that Freesurfer uses. Reading in these files are planned for a future release and may work with the functions described above. Freesurfer can also analyze diffusion tensor imaging data and some of the functions have been adapted for freesurfer but have not been thoroughly tested.\n\n\nConclusion\n\nThe neuroimaging community has developed a large collection of tools for image processing and analysis. These tools have additional functionality that is not present in R, such as the surface-based registration and processing Freesurfer provides. We have provided a similar incorporation of tools from FSL to R in the fslr package and have repeated the effort for Freesurfer with the freesurfer package to bridge this gap and provide R users functions from Freesurfer.\n\nThere has been an increasing popularity of similar interfacing of tools within the Python community such as Nipype19. As many users of R may not have experience with Python or bash scripting, we believe freesurfer provides a lower threshold for use in the R community.\n\nLowering this threshold is important because it allows more R users to control all aspects of image analysis from raw image processing to final statistical analysis. Interfacing R with existing, powerful software provides R users more functionality and a additional support community, which would not be available if the functions were rewritten in R. Although having an external software dependency may be disadvantage to R users, the software used benefits from the years of previous testing. Most importantly, as freesurfer is based on the R framework, all the benefits of using R are available, such as dynamic documents, Shiny applications, customized figures, and state-of-the-art statistical methods. This added functionality affords the neuroimaging and R communities the ability to have analysis in one framework while borrowing the strengths of both.\n\n\nExperimental features\n\nAlthough we have not thoroughly tested reading in a label file from Freesurfer, we have provided the read_fs_label function. Here we will read a label file for the left hemisphere cortex:\n\n\n\n\nSoftware availability\n\nfreesurfer is available at: https://cran.r-project.org/package=freesurfer\n\nSource code is available at: https://github.com/muschellij2/freesurfer\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.121330820\n\nSoftware license: GPL-2\n\nAll necessary code to generate this report is located at: https://github.com/muschellij2/fs_paper.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThese efforts were supported by NIH grants R01HL123407 and R01NS060910.\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 was generated using the rticles package21.\n\n\nReferences\n\nFischl B: FreeSurfer. NeuroImage. 2012; 62(2): 774–781. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSégonne F, Dale AM, Busa E, et al.: A hybrid approach to the skull stripping problem in MRI. NeuroImage. 2004; 22(3): 1060–1075. PubMed Abstract \| Publisher Full Text\n\nSled JG, Zijdenbos AP, Evans AC: A nonparametric method for automatic correction of intensity nonuniformity in MRI data. IEEE Trans Med Imaging. 1998; 17(1): 87–97. PubMed Abstract \| Publisher Full Text\n\nFischl B, Salat DH, Busa E, et al.: Whole brain segmentation: automated labeling of neuroanatomical structures in the human brain. Neuron. 2002; 33(3): 341–355. PubMed Abstract \| Publisher Full Text\n\nFischl B, Salat DH, van der Kouwe AJ, et al.: Sequence-independent segmentation of magnetic resonance images. NeuroImage. 2004; 23 Suppl 1: S69–S84. PubMed Abstract \| Publisher Full Text\n\nFischl B, Sereno MI, Tootell RB, et al.: High-resolution intersubject averaging and a coordinate system for the cortical surface. Hum Brain Mapp. 1999; 8(4): 272–284. PubMed Abstract \| Publisher Full Text\n\nReuter M, Rosas HD, Fischl B: Highly accurate inverse consistent registration: a robust approach. NeuroImage. 2010; 53(4): 1181–1196. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBordier C, Dojat M, Lafaye de Micheaux P: Temporal and spatial independent component analysis for fMRI data sets embedded in the AnalyzeFMRI R package. J Stat Softw. 2011; 44(9): 1–24. Publisher Full Text\n\nTabelow K, Polzehl J: Statistical parametric maps for functional MRI experiments in R: The package fmri. J Stat Softw. 2011; 44(11): 1–21. Publisher Full Text\n\nClayden J: RNiftyReg: Medical Image Registration Using the NiftyReg Library. Jon Clayden and based on original code by Marc Modat and Pankaj Daga. R package version 1.1.3. 2015. Reference Source\n\nFerreira da Silva A: dpmixsim: Dirichlet Process Mixture model simulation for clustering and image segmentation. R package version 0.0-8. 2012. Reference Source\n\nFeng D, Tierney L: mritc: A package for MRI tissue classification. J Stat Softw. 2011; 44(7): 1–20. Publisher Full Text\n\nJenkinson M, Beckmann CF, Behrens TE, et al.: FSL. NeuroImage. 2012; 62(2): 782–790. PubMed Abstract \| Publisher Full Text\n\nWhitcher B, Schmid VJ, Thornton A: Working with the DICOM and NIfTI Data Standards in R. J Stat Softw. 2011; 44(6): 1–28. Publisher Full Text\n\nMuschelli J, Sweeney E, Lindquist M, et al.: fslr: Connecting the FSL Software with R. R J. 2015; 7(1): 163–175. PubMed Abstract \| Free Full Text\n\nZhang Y, Brady M, Smith S: Segmentation of brain MR images through a hidden Markov random field model and the expectation-maximization algorithm. IEEE Trans Med Imaging. 2001; 20(1): 45–57. PubMed Abstract \| Publisher Full Text\n\nPau G, Fuchs F, Sklyar O, et al.: EBImage--an R package for image processing with applications to cellular phenotypes. Bioinformatics. 2010; 26(7): 979–981. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdler D, Murdoch D, et al.: rgl: 3D Visualization Using OpenGL. R package version 0.96.0. 2016. Reference Source\n\nGorgolewski K, Burns CD, Madison C, et al.: Nipype: a flexible, lightweight and extensible neuroimaging data processing framework in python. Front Neuroinform. 2011; 5: 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuschelli J, Sweeney E: muschellij2/freesurfer v1.6.2.9002 (Version v1.6.2.9002). Zenodo. 2018. Data Source\n\nAllaire JJ; R Foundation, Wickham H, et al.: rticles: Article Formats for R Markdown. R package version 0.2. 2016." }	[ { "id": "35181", "date": "06 Jul 2018", "name": "Seth T. Lirette", "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\nMuschelli, Sweeney, and Crainiceanu have provided researchers (and particularly statisticians) with a useful new tool for analyzing structural neuroimaging data. They cite a number of packages for analyzing and manipulating imaging data, but lack the functionality of Freesurfer. They well document the processes for reading and converting file formats to obtain usable R objects. The examples for plotting, bias-field correction, surface mapping, and brain extraction are clear, concise, and provide readers with enough information to recreate these with their own data. A very minor addition I would like to see would be an example of how to extract the volumetric and segmented volumetric data from a sample of subjects (or from multiple studies on the same subject). These could then be stored in other R objects (vectors, data frames, etc.) to be further analyzed using statistical methodologies. Overall, this is a very nice tool, and I personally am excited to use freesurfer very soon.\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": "35180", "date": "16 Jul 2018", "name": "Pierre Lafaye de Micheaux", "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 a very interesting R package that enables R users to have access, directly from within R, to several functions from the great neuroimaging open source software Freesurfer. The paper is well written, and their package useful.\nI only have minor comments to improve the readability of the paper.\nFirst, I would suggest to use brackets () to indicate freesurfer R functions and avoid confusion with Freesurfer command line functions. For example recon_all() for freesurfer and recon_all fro Freesurfer.\nNext, I tried to replicate all the results presented in the paper. My OS is Linux Debian 9.3 Stretch on a DELL laptop.\nFirst, I tried to install Freesurfer, following the link given in the paper. Two commands I had to use to make things work are: sudo apt-get install tcsh sudo ln -s /usr/lib/x86_64-linux-gnu/libpng16.so /usr/lib/x86_64-linux-gnu/libpng12.so.0\nThen I wanted to install FSL. I think the authors of the current paper should give a URL (e.g., https://fsl.fmrib.ox.ac.uk/fsldownloads_registration) to indicate where to download FSL. Unfortunately, FSL binaries are not available (yet) for Debian 9. I tried to install from the sources with no success. I then went back to the same URL and chose Debian 8, to download the fslinstaller.py file, then run the following command: python fslinstaller.py\nThis finally worked as expected. The authors of the current paper might want to indicate that these installation can take quite some time.\nI then launched an R session directly from the console, and typed the instructions given in the paper to check if they all work fine.\nI would suggest to give the following instructions before the user have to type all the instructions given in the paper. It was obvious for me, but might not be so for less experienced R users:\ninstall.packages(\"fslr\") install.packages(\"freesurfer\") install.packages(\"rgl\") install.packages(\"neurobase\")\nI suggest that close to this sentence \" If we were to run all the analyses, we would use the recon_all code (described below):\", the authors indicate that this is not done since it would take more than 20 hours (if this is indeed the case).\n\nI goth this first error: > L = fslr::rpi_orient(img) Error in get.fsl() : Can't find FSL In addition: Warning message: In get.fsloutput() : Can't find FSLOUTPUTTYPE, setting to NIFTI_GZ\nI then typed: > fslr::have.fsl() [1] FALSE\nI solved this error with: > options(fsl.path=\"/home/lafaye/fslbuild/fsl\") > fslr::have.fsl() [1] TRUE\nOf course, this is because, due to hard-disk space constraints, I installed FSL (and also Freesurfer) in my home directory instead of the standard location. Maybe this could be mentioned.\nWhen I typed this instruction: > nu_from_mnc = nu_correct(file = mnc) it worked but I got this warning: Malformed NIfTI - not reading NIfTI extension, use at own risk!\nWhen I typed this instruction: > nu_masked = nu_correct(file = reoriented_img, mask = mask) I got this error: Error in nu_correct(file = reoriented_img, mask = mask) :\n\nobject 'mask' not found\nIndeed, the object 'mask' is defined later on. I suggest to create this 'mask' object first, or otherwise to type the above instruction later.\nThis instruction also failed: > ortho2(ss, mask = ss) Error: could not find function \"ortho2\"\nOne should use (if the package neurobase is not loaded): > neurobase::ortho2(ss, mask = ss) > mask = ss >0\nI then retried the previous instruction now we have the mask: > nu_masked = nu_correct(file = reoriented_img, mask = mask)\nbut I got this error: Direction cosines of /tmp/RtmpxVKjYw/file29ad28e1ded7.mnc and /var/tmp/nu_correct_11320/file29ad516b90fd.mnc do not match Failed to shrink mask volume. nu_correct: crashed while running nu_estimate_np_and_em (termination status=65280) mnc2nii -float \"/tmp/RtmpxVKjYw/file29ada6796e2.mnc\"\n\n\"/tmp/RtmpxVKjYw/file29ad2a6e70b2.nii\";\nCan't find input file '/tmp/RtmpxVKjYw/file29ada6796e2.mnc' Error in mnc2nii(tmpfile, outfile = outfile) :\n\nmnc2nii did not produce outfile specified\nNot sure what to do here ...\nApart form that, everything was fine!\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/7-599
https://f1000research.com/articles/6-1827/v1	12 Oct 17	{ "type": "Study Protocol", "title": "Stage 1 Registered Report: Effect of deficient phagocytosis on neuronal survival and neurological outcome after temporary middle cerebral artery occlusion (tMCAo)", "authors": [ "Julius V. Emmrich", "Jonas J. Neher", "Philipp Boehm-Sturm", "Matthias Endres", "Ulrich Dirnagl", "Christoph Harms", "Jonas J. Neher", "Philipp Boehm-Sturm", "Matthias Endres", "Ulrich Dirnagl" ], "abstract": "Stroke is a major cause of death and disability worldwide. In addition to neuronal death resulting directly from energy depletion due to lack of blood supply, inflammation and microglial activation following ischemic brain injury has been increasingly recognized to be a key contributor to the pathophysiology of cerebrovascular disease. However, our understanding of the cross talk between the ischemic brain and the immune system is limited. Recently, we demonstrated that following focal ischemia, death of mature viable neurons can be executed through phagocytosis by microglial cells or recruited macrophages, i.e. through phagoptosis. It was shown that inhibition of phagocytic signaling pathways following endothelin-1 induced focal cerebral ischemia leads to increased neuronal survival and neurological recovery. This suggests that inhibition of specific phagocytic pathways may prevent neuronal death during cerebral ischemia. To further explore this potential therapeutic target, we propose to assess the role of phagocytosis in an established model of temporary (45min) middle cerebral artery occlusion, and to evaluate neuronal survival and neurological recovery in mice with deficient phagocytosis.", "keywords": [ "stroke", "neuroinflammation", "phagocytosis", "phagoptosis", "middle cerebral artery occlusion", "MCAo", "microglia" ], "content": "Introduction\n\nStroke is the second leading cause of death and a major cause of disability worldwide. Following a sudden interruption of the blood supply, there are two major regions of injury within the brain: the ischemic core, where severe hypoperfusion causes rapid cell death and the comparatively less hypoperfused ischemic penumbra, where cells may be stressed and dysfunctional but viable1. Within the ischemic penumbra, the fate of individual neurons results from a complex interplay of numerous biochemical and cellular events, which may lead to irreversible cell death or survival and tissue repair. Among those, activation of microglia, the primary immune effector cells of the brain, or recruited macrophages is a key feature of the pathophysiology of cerebral ischemia. Apart from their potential to release a number of pro- and anti-inflammatory and cytotoxic molecules, activated microglia demonstrate many characteristics of macrophages, including amoeboid-like morphology, capacity to migrate, antigen presentation, and phagocytic activity2,3. Consequently, one of the main effector functions of microglia is the phagocytic removal of cell debris or dying cells. In these cases phagocytosis is beneficial, because it prevents the disintegration of apoptotic cells and induces an anti-inflammatory response in microglia, thereby contributing to tissue homeostasis and repair (for review see Brown & Neher, 2014)4.\n\nMicroglial phagocytosis is closely controlled by the expression of specific cell surface ligands. For the phagocytic removal of host cells, specific ligands, so-called ‘eat-me’ signals, need to be displayed on their surface5 (Figure 1). Among a wide variety of ligands, exposure of phosphatidylserine (PS) is the best-described and most commonly observed eat-me signal. Importantly, PS externalization of neurons can occur reversibly in response to cellular stress that is not sufficient to induce cell death. In particular, stressed but viable neurons can reversibly externalize PS when exposed to non-toxic levels of oxidative or nitrosative metabolites (as may be released by microglia during inflammation)6–8, low levels of glutamate9, or growth-factor withdrawal10. In the presence of microglia this PS exposure leads to neuronal engulfment and death, whereas in the absence of microglia, these stressed neurons are able to re-internalise PS and survive6,9. Neuronal PS can be recognised by different microglial receptors, leading to a cascade of intracellular events that eventually results in the uptake of the ‘eat-me’ signal-exposing neuron11. PS can be recognised directly through microglial transmembrane receptors or indirectly through binding of soluble opsonins, such as milk fat globule EGF-like factor 8 protein (MFG-E8, also known as lactadherin or SED1)12. MFG-E8, a glycoprotein produced by microglia and astrocytes during inflammation, simultaneously engages exposed PS and the microglial vitronectin receptor (the heterodimeric integrin ανβ3/5), thereby activating phagocytosis12,13. Similar to MFG-E8, two other opsonins, growth arrest specific gene 6 (Gas6), and Protein S, which also bind to exposed PS on neurons, are in turn recognized by the membrane protein Mer receptor tyrosine kinase (MerTK)14. Of note, MerTK can also be activated downstream of the vitronectin receptor, indicating convergence of the MFG-E8 and MerTK pathways.\n\nMicroglial phagocytosis of neurons is regulated by the neuronal presentation and microglial recognition of ‘eat-me’ (left) and ‘don‘t eat-me’ (right) signals. [Figure and legend reproduced with permission from: Brown GC & Neher JJ. Microglial phagocytosis of live neurons. Nat Rev Neurosci 20144].\n\nTraditionally, phagocytosis has been regarded to occur secondary to a target cell being dead or dying. However, accumulating evidence suggests, that during neuroinflammation or cerebral ischemia phagocytes can also eat viable neurons, and thereby induce cell death (for review see Brown & Neher, 2014)4. This form of cell death resulting from the cell being phagocytosed has been termed ‘phagoptosis’15, with the deﬁning characteristic that inhibition of phagocytosis prevents cell death (Figure 2). Using a rodent model of focal cerebral ischaemia induced by stereotactic microinjection of the vasoconstrictive peptide endothelin-1 (ET-1) into the striatum or sensorimotor cortex of rats or mice, respectively, we previously found that the phagocytic proteins MFG-E8 and MerTK were transiently upregulated by microglia within the ischaemic area peaking at 3–7 days after insult. Animals deficient for MFG-E8 or the microglial phagocytic receptor MerTK had reduced brain atrophy and improved neurological function. While the number of microglial cells and the levels of inflammatory mediators were indistinguishable between genotypes, microglia from Mfge8 and Mertk knockout animals showed reduced phagocytosis of neurons9. In conclusion, these results suggest that deficiency of MerTK or MFG-E8 blocks phagocytosis of neurons by microglia and thereby prevents engulfment-induced neuronal death. However, the observed behavioural benefits among phagocytosis-deficient animals were moderate at best and the ET-1 ischemia model may have confounding effects on neuroinflammation and neuronal survival as ET-1 receptors are also expressed by neurons, astrocytes, and microglia16,17.\n\nRecent data indicate that phagocytosis can execute the death of viable neurons during development, inflammation, and neuropathology. This form of cell death is called phagoptosis, which means that cell death is caused by the cell being phagocytosed, with the defining characteristic that inhibition of phagocytosis or phagocytic signalling prevents cell death. Experimentally distinguishing between primary phagocytosis (that is, phagoptosis) and secondary phagocytosis (that is, the phagocytosis of a cell dying by apoptosis or necrosis) is possible through inhibiting phagocytosis, which in the first case will leave live cells, whereas in the second case it will leave dead cells (at least temporarily before their disintegration). [Figure and legend reproduced with permission from: Brown GC & Neher JJ.; Nat Rev Neurosci 20144].\n\nWe therefore propose to investigate how microglial phagocytosis and specific phagocytic signalling pathways contribute to the pathophysiology of stroke, by using an established model of focal cerebral ischemia. We will perform histological, biochemical, and behavioural analyses of phagocytosis-deficient wildtype mice and homozygous Mfge8 and Mertk knockout mice, and use pharmacological inhibition of the microglial MFG-E8 receptor to assess whether microglial phagocytosis is beneficial or detrimental for neuronal survival and neurological function following temporary (45min) middle cerebral artery occlusion (tMCAo). In these animals, we will test:\n\n1) Whether phagocytic deficiency is beneficial or detrimental for neurological function; and\n\n2) Whether phagocytic microglia and recruited macrophages contribute to neuronal and/or synaptic loss following cerebral ischemia and if this is beneficial or detrimental for tissue recovery.\n\nBy pre-registering this study we strive to foster transparency about our aims, study design, and analysis plan, thereby strengthening the robustness and accountability of our data.\n\n\nMethods\n\nAll animal experiments will be performed in accordance with local regulations, and have been approved by the Berlin governmental authorities (Landesamt für Gesundtheit und Soziales, LaGeSo), approval number G057/16.\n\nMale C57BL/6NCrl mice will be derived from Charles River at the age of 8 weeks. Phagocytosis-deficient Mertk (Jax: B6;129-Mertktm1Grl/J) and Mfge8 (from C. Théry, INSERM 932, France)18 knockout mice will be derived from The Jackson Laboratory and Hertie Institute for Clinical Brain Research, respectively, and bred locally. Male homozygous Mertk and Mfge8 knockout mice and their homozygous wildtype littermates will be used in experiments at the age of 10 – 12 weeks. Animals will be group-housed with ad libitum access to food and water and cages will be equipped with environmental enrichment tools (red transparent plastic nest box and brown paper towels). Animals will be kept in specific pathogen free (SPF) conditions under a 12 h light/dark cycle (lights on: 8am; lights off: 8pm). Room temperature will be maintained at 22 ± 1°C.\n\nAnimals will be randomized using the GraphPad calculator tool (http://www.graphpad.com/quickcalcs/randomize1.cfm) by a researcher who is not involved in the surgical procedure, behavioral, histological, biochemical or MRI analysis. Information on genotype and treatment group assignment will be concealed from experimenters until the end of the study. Behavioral, histological, biochemical and MRI analysis will be performed by researchers who are not involved in the surgical procedure.\n\nAnimals will be excluded from this study if: i) infarct volume is not detected and/or non-middle cerebral artery territory ischemia (such as cerebellar infarction) is detected on MRI imaging at 24h following cerebral ischemia; ii) pellet-reaching performance in the Staircase test differs by more than 1.2 standard deviations from the mean performance of the corresponding genotype and the treatment group at the end of the conditioning phase.\n\nAll animals will be induced with 2% isoflurane in oxygen and nitrous oxide (ratio 0.3/0.7) and maintained with 1% isoflurane during surgical or imaging procedures.\n\nTemperature will be maintained at 37 ± 0,5°C throughout the surgery or imaging procedures using a heating blanket. In addition, following cerebral ischemia body temperature and weight will be measured once daily. Temperature measurements will be obtained non-invasively using subcutaneous radio-frequency identification (RFID)-transponders (IPTT-300; Bio Medic Data Systems). Transponders will be implanted by subcutaneous injection at least one week prior to the induction of cerebral ischemia.\n\nMice will be subjected to 45 minutes filamentous temporary middle cerebral artery occlusion (tMCAo), and will be killed for histological and biochemical analyses after 3, 5, or 28 days, respectively. The filamentous tMCAo model will be performed using Dirnagl et al ’s method (doi: 10.1038/npre.2010.3492.2) as implemented in our laboratory19–21. For pain relief, Bupivacaine gel is topically applied in the wound, and the wound is temporally closed with an adaptive suture.\n\nFor inhibition of phagocytic signaling mice will be treated with EMD121974 (Cilengitide®, Selleckchem Inc.), a commercially available vitronectin receptor-antagonist. EMD121974 or control (PBS, 10 ml/kg) will be administered daily by intraperitoneal injection for 7 days after tMCAo. The first dose of EMD121974 (30 mg/kg) is administered intraperitoneally 6 h after tMCAo, followed by daily doses of 10 mg/kg.\n\nMagnetic resonance imaging (MRI) will be performed at 24 h and 21 days following tMCAo using a 7 Tesla rodent scanner (BioSpec 70/16AS, Bruker BioSpin, Ettlingen, Germany) with a 16 cm horizontal bore magnet and a 9 cm (inner diameter) shielded gradient with an H-resonance frequency of 300 MHz and a maximum gradient strength of 300 mT/m. For imaging, a quadrature volume resonator with an inner diameter of 86 mm for excitation and a decoupled mouse head quadrature surface coil for signal reception (both Bruker) will be used. Data acquisition and image processing will be performed with the Bruker software Paravision 6.0.1. and custom MATLAB (MathWorks, Natick, MA, USA) scripts. Both, at 24 h and 21 d, a rapid acquisition with relaxation enhancement (RARE) T2-weighted (T2w) sequence will be used for anatomical imaging (repetition time TR=3500, echo time TE=33 ms, RARE factor 8, 4 averages, 32 contiguous coronal slices, slice thickness 0.5 mm, field of view FOV=1.92 × 1.92 cm2, matrix MTX=192×192, total acquisition time TA=5:36 min). At 21 d, in addition to anatomical imaging, diffusion tensor imaging (DTI) using a Stejskal-Tanner 4-shot spin echo EPI pulse sequence will be performed (TR/TE=3500 ms/28.5 ms, 1 average, geometry identical to the T2w image, MTX=96×96, high angular resolution diffusion (HARDI) encoding scheme with 60 diffusion directions, b=1000 s/mm2, five b=0 images, diffusion duration δ=2.6 ms, diffusion separation Δ=8.5 ms, TA=15:10 min). Maps of fractional anisotropy and mean/axial/radial diffusivity (FA, MD, AD, RD) will be calculated and the DTI connectome of each mouse will be reconstructed using whole brain fiber tracking in DSI studio (http://dsi-studio.labsolver.org/) as described previously22. The stroke lesion will be segmented on T2w images 24 h post-surgery using Analyze 10.0 software (AnalyzeDirect, Overland Park, KS, USA) by selecting hyperintense areas of ischemic tissue by an experienced researcher and a mask will be exported. The 24 h lesion mask, and 21 d DTI parameter maps will coregistered to the Allen mouse brain atlas using their corresponding anatomical T2w images in the MATLAB toolbox ANTX (https://github.com/philippboehmsturm/ANTX/)23–25. Since the registration includes nonlinear terms, lesion volumes are effectively corrected for edema when measured in atlas space. Edema-corrected lesion volume and DTI parameters will be measured in all regions of the atlas and in a volume of interest encompassing the lesion territory at 24 h. Values will be compared using two sample t-test using false discovery rate post-hoc correction. ANTX will also be used to coregister a modified atlas with less brain structures (MRMNeAt) to the DTI connectome and a graph theoretical analysis will be performed using the brain connectivity toolbox followed by comparison of scalar network parameters as described previously22,26.\n\nDeSimoni’s neuroscore27, a composite of general behavioral alterations and focal motor, sensory, reflex, and balance deficits, will be performed at days 1, 2, 7, 14 and 21 following cerebral ischemia as implemented in our laboratory19. In brief, general health and behavioral alterations and specific focal deficits will be scored separately and subsequently added to form a summation score. The maximum score is 43 points, with more points meaning more deficits.\n\nThe Rotarod, a widely used test of motor coordination and learning, will be performed at days 2, 7, and 14 following cerebral ischemia as implemented in our laboratory19. The best run of three runs per time point will be used for statistical analysis.\n\nThe Staircase test will be performed daily for 7 days before and up to 21 days after cerebral ischemia for conditioning and testing skilled forelimb motor function, respectively. Mice will be placed in a Plexiglas holding box with an attached baited double staircase. Food rewards are presented bilaterally on a descending double staircase with each of the 8 steps containing a food-well loaded with 20 mg sucrose pellets (Sandown Scientific). During testing, the animal is required to ascend onto a central base with one staircase on either side, designed such that only the ipsilateral paw can reach a given staircase. A lip on the platform edge prohibits the animal from dragging the pellet up alongside the platform, thus requiring it to be grasped and brought up and around the platform edge. The pellets in lower wells are more difficult to grasp than those in wells higher on the staircase, thus providing objective measures of maximum forelimb extension and grasping skill. Outcome is measured as the number of pellets grasped and eaten with each forepaw. Animals are tested once daily for 20 minutes per session.\n\nFollowing perfusion with physiological saline and 4% paraformaldehyde (PFA) and cryosectioning of the fixed tissue, brain sections will be stained for cresylviolet or NeuN to label all surviving cells and infarct size will be determined by stereological quantification by a blinded observer on random sets of every 12th systematically sampled 40 μm thick sections throughout the brain. Analysis will be conducted using the Stereologer software (Stereo Investigator 6; MBF Bioscience) and a motorized x-y-z stage coupled to a video microscopy system (Optronics) as previously described28, with application of the Cavalieri estimator technique29. Neuronal and microglial densities will be quantified using the optical fraction fractionator on sections stained for NeuN and ionized calcium binding adaptor molecule 1 (Iba1), respectively30. To quantify microglial phagocytosis of neurons, high-resolution confocal z-stack images of the peri-infarct area stained for microglia (Iba1) and neuronal nuclear antigen (NeuN) will be obtained. Z-stack acquisition will be followed by 3-dimensional reconstruction using Imaris software (Bitplane, UK) and quantifying the percentage of microglia that contain NeuN-positive inclusions (as described in detail in 9).\n\nThe study is designed with 80% power to detect a relative 25% difference in pellet-reaching performance in the Staircase test. A priori power analysis using a repeated measures ANOVA with Tukey’s post hoc test under the following assumptions α = 0.05, β = 0.2, mean, SD 20% of the mean determines the number of required experimental units at 17 animals per group. Based on previous results with this model we estimate that 15% of animals will have to be excluded from this study based on insufficient infarct volume (10%) or insufficient pellet-reaching performance in the Staircase test, respectively. Thus, we will use 20 animals per genotype or treatment group, respectively (Mertk and Mfge8 phagocytosis-deficient knockout animals vs. littermate controls and phagocytosis inhibitor-treated animals vs. untreated controls, respectively). Data will be tested for normal distribution using D’Agostino’s K2 test and analyzed by 1-way analysis of variance (ANOVA) or in case of measuring the effects of 2 factors 2-way ANOVA with posthoc Holm-Sidak adjustment for p values. For DTI parameter maps, lesion volumes, and network analysis metrics a false discovery rate to control for multiple comparisons and Type I errors will be used. If only 2 groups are compared, unpaired, two-tailed Student’s t test will be used. Nonparametric functional data will be compared using Kruskal-Wallis test with posthoc Dunn multiple comparison test. Survival will be compared for the effect of genotype or treatment using a log-rank Mantel–Cox test. P values ≤ 0.05 are considered statistically significant.\n\nFunctional, MRI, and histological outcome data of phagocytosis-deficient Mertk and Mfge8 knockout animals and littermate controls will be assessed using a block design (i.e. all animals will be tested before data analysis is performed). Outcome data of phagocytosis inhibitor-treated animals vs. untreated controls will be assessed using a group sequential design with 4 stages (k=4, sample size at stage 1, 2, and 3, respectively: 14 animals (7 animals vs. 7 animals), sample size at stage 4: 12 animals (6 animals vs. 6 animals), for details see 31). To save animals and money, testing will be terminated following unblinded interim data analysis at each stage based on the following stopping criteria: stage 1) none or detrimental effect of phagocytosis inhibitor on neuronal and/or synaptic survival and tissue recovery at day 5 following tMCAo; stages 2–4) detrimental or beneficial effect of phagocytosis inhibitor on pellet-reaching performance in the Staircase test following tMCAo (i.e. mean pellet-reaching performance differs by more than 20% from one standard deviation of the mean pellet-reaching performance of the control group).\n\nThe study will be conducted within 12 months, following successful peer review of this Stage 1 Registered Report submission.", "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\nMari C, Karabiyikoglu M, Goris ML, et al.: Detection of focal hypoxic-ischemic injury and neuronal stress in a rodent model of unilateral MCA occlusion/reperfusion using radiolabeled annexin V. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "26908", "date": "18 Oct 2017", "name": "Stuart M. Allan", "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 rationale behind the study is well explained and on the most part the methods are described in full. Importantly the authors provide full details of experimental design, including power calculations and planned statistical analyses. They also propose the use of sequential design which is very unusual in preclinical research, but can be used more widely.\n\nOverall there are no major concerns regarding the planned study but the report would benefit from additional detail and clarification in a few places as detailed below:\nThe authors do not specify what will be their primary endpoint and what will constitute secondary analyses. The study is powered on the Staircase test which would imply that this is being taken as the primary outcome. The title and abstract should be amended to clarify this, and the latter should also have details of the secondary outcomes that are being assessed.\n\nThe abstract would benefit from additional details on the age and sex of mice used.\n\nPage 2: The authors indicate no grants were involved in supporting the work. It is possible to clarify therefore exactly how the work was supported, since presumably the funds had to come from somewhere.\n\nPage 5: Clarify if a homeothermic feedback system used to maintain body temperature.\n\nPage 5: Clarify if modified diet provided to aid post-stroke recovery and on similar lines if fluid replacement instigated.\n\nPage 5: Clarify why systemic analgesia not used.\n\nPage 5: Clarify how dosing regimen decided on.\n\nPage 5: Clarify why only male mice being used.\n\nPage 6: For rotarod clarify why best run being used rather than average.\n\nPage 6: Please specify endpoints (days post MCAo) when animals are perfused.\n\nPage 7: day 5 has not been previously specified as an endpoint.\n\nHave the authors pre-specified sufficient outcome-neutral tests for ensuring that the results obtained can test the stated hypotheses, including positive controls and quality checks? Yes\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? Yes", "responses": [ { "c_id": "3173", "date": "14 Nov 2017", "name": "Julius Emmrich", "role": "Author Response", "response": "We would like to thank the reviewer for the insightful and constructive remarks. All points have also been addressed in the revised version of this article. Point 1: The primary outcome measure of this study is functional recovery following tMCAo as determined with the staircase test. The study has been powered accordingly. Secondary outcomes constitute Rotarod performance, stroke volume (quantified on MR imaging or brain sections, respectively), diffusion tensor imaging (DTI) connectome mapping, and histological analyses to measure neuronal and microglial densities, and phagocytic activity. Point 2: Male mice at the age of 10-12 weeks will be used for experiments. The abstract has been modified accordingly. Point 3: Funds for transgenic animal breeding will be provided by the German Federal Institute for Risk Assessment and parts of the study will be supported by grants of the Federal Ministry of Education and Research (BMBF) for the Center for Stroke Research Berlin to UD, CH and PBS and the Berlin Institute of Health, QUEST Center to UD. Point 4: Temperature will be maintained at 37 ± 0,5 °C during surgery or imaging procedures using a closed loop homeothermic feedback system which consist of a heating blanket connected to a rectal probe. Point 5: Following tMCAo animals will have access to mashed chow placed on the cage floor in a petri dish for up to 24 hours after surgery. There will be no additional fluid replacement. Point 6: The brain itself is not sensitive to pain. Local delivery of gel comprising anesthetics is sufficient to reduce the pain that originates from the wound and the vessels. Many inflammatory signals are modulated by non-steroid analgesic drugs or opioids. Therefore, we aimed to avoid using drugs that probably affect stroke outcome or inflammatory signaling including phagoptosis. Point 7: The dosing regimen of EMD121947 (cilengitide), an inhibitor of phagocytic signaling, was decided based on results from previous pharmacokinetic studies in mice and preliminary, unpublished data. Point 8: Only male mice will be included in this study, which might introduce a potential sex bias. However, including female animals is likely to increase variation, which, in turn, would require the use of additional animals, time, and effort. The number of subjects for this study was limited to the minimum required to obtain scientifically meaningful results and only the use of male mice has been approved by the Berlin Ethical Review Panel (LaGeSo). If results from the pharmacological intervention study will be encouraging, we would consider planning a pre-clinical trial that is powered to study sex differences by EMD121947. Point 9: We expect that the variation of Rotarod results will be smaller if the best run out of three trials is used rather than the average. If we would calculate averages across three trials, an accidental slip or drop off the rod that is caused by lack of attention or motivation would falsely increase variation. We believe that this approach is best suited to obtain adequate information on the motor coordination of a given animal. Point 10: As specified on page 5 in the Materials and Methods section, endpoints will be at 3, 5, or 28 days following 45min tMCAo, respectively. Point 11: As specified on page 5 in the Materials and Methods section, animals will be killed for histological and biochemical analyses after 3, 5, or 28 days after 45min tMCAo, respectively. To improve clarity, this information has now also been included on page 7 (study design)." } ] }, { "id": "26912", "date": "31 Oct 2017", "name": "Ádám Dénes", "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 find it a great idea to share the design of the proposed studies with the scientific community. In general, the aim of the research is great and the methods to be used are largely appropriate. The authors deserve the credit for providing details on given experimental objectives, including randomisation, blinding, power calculation and desrciption of the experimental procedures, which all increase the transparency and credibility of these studies.\nSpecific points:\n1. It is stated that the studies will be completed in 12 months, which is doable given that all mouse strains are available and breeding on site. Based on what is described in the methods, it remains unclear how long it will take to establish breeding colonies from the phagocytosis-deficient strains derived from The Jackson Laboratory and how the phenotype of these will be compared to the control animals, which will be obtained from another vendor (Charles River). For such studies comparing different KO lines, it would be useful to ensure the identitiy of the background ideally by establishing heterozygous breeding colonies and use littermates for the experiments. C57BL/6 (and other) mouse stains from different vendors could show markedly different inflammatory or phagocytic responses that could influence the interpretation of the results.\n2. In addition to using Mfge8 KO mice, the pharmacological inhibition of the microglial MFG-E8 receptor is also proposed in the methods. If phagocytic signaling will be blocked by intraperitoneal administration of the antagonist, will this regimen effectively reach areas of the brain where there is no major BBB injury? Details on brain penetration and pharmacokinetics of the drug (if known) would be useful.\n3. The phagocytosis-deficient lines and pharmacological inhibitors will likely change phagocytic responses in all tissue macrophages, including perivascular macrophages in the brain (the latter will possibly be influenced by the pharmacological treatment earlier than microglia). Unless the authors plan sophisticated studies on descriminating microglial actions from that of blood-borne phagocytes that may be recruited into the affected brain areas, it may be mose useful to talk about microglia /macrophages throughout. Iba1 is not an ideal marker either to discriminate brain microglia /macrophages of different origin.\n4. Several excellent sensory-motor tests are planned for functional assessment, among which the staircase requires extensive conditioning and testing. Has the same battery of tests been successfully used in similar MCAo studies previously? Extensive testing might lead to overtraining of mice and a reduction of differences in performance between experimental groups in spite of the expected changes in the number of surviving neurons.\nThe notes above merely aim to help the interpretation of these excellent studies rather than suggesting extensive changes in experimental design, particularly regarding costly breeding and determination of microglial-specific effects.\nHave the authors pre-specified sufficient outcome-neutral tests for ensuring that the results obtained can test the stated hypotheses, including positive controls and quality checks?\nYes.\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? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "3172", "date": "14 Nov 2017", "name": "Julius Emmrich", "role": "Author Response", "response": "We would like to thank the reviewer for appreciating the aim of this registered report and the helpful comments. Where appropriate the manuscript has been revised following the reviewer’s remarks. Point 1: We agree with the reviewer that the description of breeding procedures lacked some detail. Heterozygous breeding colonies for phagocytosis-deficient mouse strains have already been established in our laboratory. Homozygous Mertk and Mfge8 knockout offspring will be used in experiments. Homozygous wildtype littermates will serve as controls. C57BL/6NCrl mice, which will be obtained from Charles River, will be used for inhibition experiments. Point 2: Several pharmacokinetic studies have shown penetration of EMD121947 (cilengitide) through the intact blood brain barrier. An excellent study on the pharmacokinetics of cilengitide in mice and men by Dolgos et al. can be found here: www.ncbi.nlm.nih.gov/pmc/articles/PMC4804314. Point 3: We thank the reviewer for bringing up this important point. We do not plan to discern effects of deficient phagocytosis between microglia and blood-derived macrophages. As suggested by the reviewer, we have modified the wording throughout the manuscript. Point 4: We appreciate the reviewer’s concern that excessive training might have a negative impact on behavioral outcomes. Both tests have been established in our laboratory and have been used in parallel for MCAo studies. Several other groups have also used a combination of these tests following MCAo in mice (see for example: http://www.jneurosci.org/content/24/27/6209). It is well conceivable that training in one test might have an influence on the results of the other. The staircase and Rotarod tests assess skilled forelimb function (i.e. number of pellets grasped and eaten with each forepaw) and general motor coordination, respectively. Thus, we believe that the additional information which is gained by assessing two distinctly different motor skills outweighs a potential confounding effect." } ] } ]	1	https://f1000research.com/articles/6-1827
https://f1000research.com/articles/7-595/v1	15 May 18	{ "type": "Method Article", "title": "The use of PrP transgenic Drosophila to replace and reduce vertebrate hosts in the bioassay of mammalian prion infectivity", "authors": [ "Alana M. Thackray", "Olivier Andréoletti", "Raymond Bujdoso", "Alana M. Thackray", "Olivier Andréoletti" ], "abstract": "Prion diseases are fatal neurodegenerative conditions of humans and vertebrate species. The transmissible prion agent is a novel infectious particle composed principally of PrPSc, an abnormal isomer of the normal host protein PrPC. The only reliable method to detect mammalian prion infectivity is by bioassay, invariably in a vertebrate host. The current prion bioassays typically involve intracerebral or peripheral inoculation of test material into the experimental host and subsequent euthanasia when clinical signs of terminal prion disease become evident. It may be months or years before the onset of clinical disease becomes evident and a pre-determined clinical end-point is reached. Consequently, bioassay of prion infectivity in vertebrate species is cumbersome, time consuming, expensive, and increasingly open to ethical debate because these animals are subjected to terminal neurodegenerative disease. Prions are a significant risk to public health through the potential for zoonotic transmission of animal prion diseases. Attention has focussed on the measurement of prion infectivity in different tissues and blood from prion-infected individuals in order to determine the distribution of infectious prions in diseased hosts. New animal models are required in order to replace or reduce, where possible, the dependency on the use of vertebrate species, including the ‘gold standard’ mouse prion bioassay, to assess prion infectivity levels. Here we highlight the development of a Drosophila-based prion bioassay, a highly sensitive and rapid invertebrate animal system that can efficiently detect mammalian prions. This novel invertebrate model system will be of considerable interest to biologists who perform prion bioassays as it will promote reduction and replacement in the number of sentient animals currently used for this purpose. This article is a composite of previous methods that provides an overview of the methodology of the model and discusses the experimental data to promote its viability for use instead of more sentient hosts.", "keywords": [ "Prion", "infectivity", "bioassay", "invertebrate", "Drosophila" ], "content": "\n\n\n\nScientific benefits:\n\nDisplays higher levels of sensitivity than gold standard mouse bioassay\n\n3Rs benefits:\n\nCan replace the use of rodent and other vertebrate species used in bioassays for mammalian prion infectivity\n\nPractical benefits:\n\nBioassay can be completed within approximately 6 weeks\n\nCurrent applications:\n\nAssessment of mammalian prion infectivity\n\nPotential future applications:\n\nUnderstanding the transmissibility of prion and prion-like proteins\n\n\nIntroduction\n\nPrion diseases, or transmissible spongiform encephalopathies (TSEs) are fatal, neurodegenerative conditions of humans and various vertebrate species (Prusiner, 2004). These conditions include Creutzfeldt-Jakob disease (CJD) of humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and Chronic Wasting Disease (CWD) of cervids. Prion diseases are a significant risk to public health because of their potential for zoonotic transmission, as evidenced by the BSE epizootic in UK cattle and subsequent appearance of variant CJD in humans (Bruce et al., 1997). The emergence of new prion diseases, such as atypical scrapie in sheep (Benestad et al., 2003) and atypical BSE in cattle (Biacabe et al., 2004; Casalone et al., 2004), and new reservoirs of CWD in cervids (Benestad et al., 2016), pose fresh challenges to human food safety since their zoonotic potential is unknown. Consequently, much attention has been focussed on the detection of mammalian prion infectivity and its distribution in hosts affected by prion diseases. The goal of such studies is to understand the biology of infectious prions in order to alleviate their burden on animal health and to protect human health.\n\nIn contrast to conventional pathogens such as viruses and bacteria, prions lack a nucleic acid-based genome. Instead, the infectious prion agent comprises principally, if not solely, of PrPSc, a disease conformer of the normal host protein PrPC (Prusiner, 1982). For these reasons prions are not detected by common molecular biology techniques, such as PCR. The only reliable method to measure prion infectivity is through bioassay in experimental hosts and various mammalian animal species have been used for this purpose. Prion infectivity studies in large experimental animals, such as primates, sheep and goats, have been instrumental in establishing core features of mammalian prion biology including disease transmissibility and the existence of different prion strains in a single PrP polypeptide (Gajdusek et al., 1966; Gajdusek et al., 1968; Kimberlin, 1977; Kimberlin, 1982; Stamp, 1962). Cattle and cervids have been used for BSE and CWD pathogenesis in their natural hosts, respectively, in order to provide important information on the distribution of prion infectivity and the mechanisms of its spread in these ruminant species (Mathiason et al., 2006; Wells et al., 1998).\n\nPrion infectivity studies in large experimental hosts are hampered by long incubation times for the onset of clinical disease and the low numbers of animals used as a consequence of the difficulties in their housing, with resultant loss of statistical power. More robust and reproducible prion infectivity measurements were achieved with the discovery that sheep scrapie was experimentally transmissible to rodents (Chandler, 1961; Zlotnik & Rennie, 1965), which allowed larger numbers of experimental animals to be used. Accordingly, mice, either wild type or those transgenic for PrP autologous to the species form of prions under study, hamsters, and bank vole have collectively been used for measurement of prion infectivity from many different TSE-affected hosts including humans (Brandner & Jaunmuktane, 2017; Watts & Prusiner, 2014). However, even experimentally inoculated mice may take many months or years to develop prion disease and reach a pre-determined clinical end-point. Collectively, the bioassay of prion infectivity in vertebrate species is cumbersome, time consuming, financially very expensive, and increasingly open to ethical debate because these animals are subjected to terminal neurodegenerative disease.\n\nIn order to advance the principles of the 3Rs, namely replacement, reduction and refinement, with respect to animal experimentation in prion research, alternative methods to assess prion infectivity would be of significant benefit. Presently, in vitro cell culture systems do not exist that can detect natural isolates of important animal prion diseases such as BSE (Oelschlegel et al., 2015). Furthermore, while the in vitro amplification technique of protein misfolding cyclic amplification (PMCA) (Saborio et al., 2001) or QuIC (Atarashi et al., 2007) demonstrate the presence of abnormal PrP, they do not detect prion infectivity, which is only revealed by bioassay in an animal host. Since transmissibility is a defining hallmark of prion diseases, it is important to develop a reasonably rapid and versatile confirmatory prion infectivity bioassay to supplement in vitro biochemical-based prion diagnostic assays. The new prion bioassay is required to be as sensitive as the ‘gold standard’ mouse prion bioassay but preferably using a less sentient host and one that is less costly.\n\nHere we present methodology and experimental data that describe the use of PrP transgenic Drosophila as a viable alternative to the employment of more sentient hosts to assess mammalian prion infectivity. Our methodology has allowed us to demonstrate that the Drosophila-based prion bioassay is extremely sensitive and can detect a ≥10-10-fold dilution of scrapie-infected sheep brain homogenate (Thackray et al., 2016), a significantly higher level of sensitivity compared to the ‘gold standard’ mouse prion bioassay (Andreoletti et al., 2011). Furthermore, our fly-based prion bioassay can be completed within ≈6 weeks, in contrast to vertebrate species that may require months or years to assess the same prion inocula. In addition, we have shown that PrP transgenic Drosophila can detect prion-infected blood from asymptomatic scrapie-infected sheep (Thackray et al., 2016). This suggests this novel invertebrate system has significant practical use as a potential confirmatory blood test for prion diseased individuals, for example humans with vCJD.\n\nThis article is a composite of previously published methods to highlight the development of PrP transgenic Drosophila for use as a new prion bioassay for the sensitive and rapid assessment of mammalian prion infectivity. In doing so, we stress the utility of Drosophila to model transmissible mammalian prion disease. This new animal model will be of considerable interest to experimentalists who perform prion bioassays as it will allow reduction and partial replacement, where possible, in the number of sentient hosts currently used for this purpose.\n\n\nMethods\n\nOverview. We have generated Drosophila transgenic for topological variants of mature length ovine PrP by pUAST / PhiC31-mediated site-directed mutagenesis. These fly lines, generated by Bestgene (California, USA), were transgenic for ovine PrP together with an N-terminal leader peptide and a C-terminal GPI signal sequence [PrP(GPI)] (Thackray et al., 2012c; Thackray et al., 2014a), or expressed ovine PrP without either an N-terminal leader peptide or a C-terminal GPI signal sequence [PrP(cyt)] (Thackray et al., 2014b). PCR and DNA sequencing was used in order to confirm that each PrP transgene was present as a single copy and located at the single 51D-site in the fly genome. We subsequently removed the red fluorescent protein (RFP) cassette located at the 51D site of the fly genome by Cre-mediated cleavage in each PrP fly line. Confocal microscopy of Drosophila S2 cells transiently transfected with the different pUAST-VRQ variants showed that PrP(GPI) and PrP(ΔGPI) entered the secretory pathway, whereas PrP(cyt) was restricted to the cytosol (Thackray et al., 2014a). Non-RFP UAS-PrP transgenic fly lines were subsequently crossed with GAL4-driver lines to allow expression of PrP in Drosophila.\n\nPrP transgenic Drosophila were exposed to mammalian prions at the larval stage (Thackray et al., 2012b; Thackray et al., 2014a; Thackray et al., 2014b; Thackray et al., 2016). After hatching, Drosophila were transferred to prion-free culture tubes. At various time points during their adult lifespan, groups of Drosophila were analysed for locomotor ability, or euthanised, decapitated and homogenate prepared from the isolated fly heads (Thackray et al., 2012b; Thackray et al., 2014a; Thackray et al., 2014b; Thackray et al., 2016). These homogenates were used to seed in vitro PMCA reactions in order to reveal the presence of prion seeding activity (Thackray et al., 2014a); SDS/PAGE western blot detection of PrPSc; RNASeq-based transcriptome analysis (Bujdoso et al., 2015); or used in fly-to-fly or fly-to-mouse prion transmission studies (Thackray et al., 2016).\n\nFly stocks. The following fly lines were obtained from the Department of Genetics, University of Cambridge, UK.\n\n- Actin-5C-GAL4 (y w; P{w[+mC]=Act5C-Gal4}25F01/CyO, y[+])\n\n- Elav-GAL4 (P{w[+mW.hs]=GawB}elav[C155])\n\n- 51D (w; M{3xP3-RFP.attP}ZH-51D)\n\nPrimary transmission of sheep scrapie (sheep-to-fly). Drosophila at the larval stage of development were exposed to brain homogenate of cerebral cortex tissue from a confirmed VRQ/VRQ PG127 (alternatively referred to as DAW or G338) scrapie-positive sheep (SE1848/0005) (Thackray et al., 2008) or blood plasma from scrapie-positive sheep (Lacroux et al., 2012; Thackray et al., 2016). New Zealand-derived VRQ/VRQ scrapie-free brain tissue or blood plasma were used as control material. Two hundred and fifty microlitres of either 10% (v/v) blood plasma or 1% (w/v) of sheep brain homogenate, or a 1/10 dilution series (v/v) of these samples, prepared in PBS pH7.4, were added to the top of the cornmeal that contained third instar Drosophila larvae in 3-inch plastic vials. Following eclosion (i.e. hatching) flies were transferred to fresh non-treated vials.\n\nSecondary transmission of sheep scrapie (fly-to-fly). Drosophila head homogenates were prepared from 30 day old flies that had been exposed at the larval stage to scrapie-positive or scrapie-negative sheep brain material. Two hundred and fifty microlitres of a 10-1 (v/v) dilution of the original fly brain homogenate were added to the top of the cornmeal that contained third instar Drosophila larvae in 3-inch plastic vials. In all cases, flies were transferred to fresh, non-treated vials following eclosion.\n\nPreparation of Drosophila head homogenate. Whole flies in an eppendorf tube were frozen in liquid nitrogen for 10 minutes and then vortexed for 2 minutes to cause decapitation. Individual fly heads were isolated and placed in clean eppendorf tubes using a fine paint brush. PBS pH 7.4 was added to give 1µL / head and homogenates were prepared by manual grinding of the fly heads with sterilised plastic pestles. For western blot analysis, fly head homogenate was mixed with an equal volume of 20% scrapie-free sheep brain homogenate prior to extraction and PK digestion as previously described (Lacroux et al., 2012) using monoclonal antibody Sha31 (Feraudet et al., 2005).\n\nProtein misfolding cyclic amplification (PMCA). PMCA was carried out as previously described (Lacroux et al., 2012). The substrate consisted of 10% (w/v) ovine VRQ PrP (tg338) transgenic mouse brain homogenate in PBS pH 7.4, 0.1% Triton X-100 and 150 mM NaCl buffer (Lacroux et al., 2014). Five µL of fly head homogenate were mixed with 45µL of substrate in 0.2 mL thin wall PCR tubes. Sealed tubes were then placed in the horn of a Misonix 4000 sonicator for one round of 96 cycles. Each cycle consisted of a 10 second sonication step (70% of power) followed by a 14 minute and 50 second incubation step. Twenty µL of each reaction mix were subsequently treated with PK (4µ g of PK per mg of protein) at 37°C for 2 hours and the reaction stopped by adding Pefabloc (4mM final concentration). PK-resistant PrP was detected by western blot as previously described (Lacroux et al., 2012) using monoclonal antibody Sha31 (Feraudet et al., 2005).\n\nFly-to-mouse prion transmission. Fly-to-mouse prion transmission was carried out in ovine VRQ PrP (tg338) transgenic mice (Le Dur et al., 2005), which are highly efficient for the detection of ovine prion infectivity. All mouse bioassays were performed under licence number D-31-555-27, in compliance with institutional and national guidelines including ethical approval, and in accordance with the protection of animals used for scientific purposes under European Community Council Directive 2010/63/UE. Female tg338 mice (n=6) bred in-house aged 12 – 14 weeks were housed in a single cage with environmental enrichment and maintained under controlled conditions with respect to lighting, temperature, humidity and noise. Mice were injected intracerebrally with 20µL of diluted fly head homogenate (to give approximately 2 fly head equivalents per mouse) and monitored daily until the occurrence of clinical signs of mouse prion disease. Inoculated mice were euthanised when they started to show locomotor disorders and any impairment in their capacity to feed, or at a pre-defined end-point for the assay (>250 days) (Andreoletti et al., 2011). Brain tissue (cerebral cortex) was collected from euthanised mice and frozen for PrPSc analysis by Western blot (TeSeE, BioRad) or PET blot analysis (Andreoletti et al., 2011).\n\nThe locomotor ability of flies was assessed in a negative geotaxis climbing assay initiated with 45 (3 × n=15) age-matched, pre-mated female flies in each treatment group (Nichols et al., 2012; Thackray et al., 2014a; Thackray et al., 2014b). Drosophila were placed in adapted plastic 25mL pipettes that were used as vertical climbing columns and allowed to acclimatise for 30 minutes prior to assessment of their locomotor ability. Flies were tapped to the bottom of the pipette (using the same number and intensity of taps on each occasion) and then allowed to climb for 45 seconds. At the end of the climbing period the number of flies above the 25mL mark, the number below the 2mL mark and the number in between the 2mL and 25mL mark was recorded. This procedure was performed three times at each time point. The performance index (PI) was calculated for each group of 15 flies (average of 3 trials) using the formula: PI = 0.5 × (ntotal + ntop – nbottom)/ntotal where ntotal is the total number of flies, ntop is the total number of flies at the top, and nbottom is the total number of flies at the bottom. A PI value of 1 is recorded if all flies climb to the top of the tube whereas the value is 0 if no flies climb the tube past the 2mL mark. The mean PI ± SD at individual time points for each treatment group was plotted as a regression line.\n\nDetailed methodology of the climbing assay is as follows:\n\nPreparation of climbing assay pipettes\n\nPlastic 25mL pipettes used in the climbing assay were prepared by taking a sharp saw blade and carefully cutting the top off the pipette. The cut edges were filed down in order to prevent damage to the Drosophila wings when the flies were added to, or removed from, the pipettes before or after the climbing assay was carried out. The tip of each pipette was sealed with a small piece of nescofilm wrapped securely around the point in order to prevent the escape of Drosophila during the assay. Clean cotton wool plugs were pushed into the top of each pipette to ensure a close fit so that the Drosophila could not climb out of the pipette once the assay had started.\n\nAddition of flies to the climbing assay pipettes\n\nAt the start of each assay, the flies were counted in each set of fly vials dedicated to each treatment group in order to verify the number present (typically 3 vials, each containing 15 flies at the start of the experiment). The Drosophila from one vial were gently tipped into the top of a pipette using a dedicated plastic funnel for each treatment group. The cotton wool plug was securely fitted to stopper the top of the pipette as soon as the funnel was removed. The Drosophila were tapped to the bottom of the pipette, which was then laid horizontal and the flies allowed to acclimatise prior to the assay.\n\nAcclimatisation of flies in the climbing assay pipettes\n\nThe pipettes that contained the Drosophila were placed horizontal at 25°C for 30 minutes in order to allow the flies to acclimatise. After the acclimatisation period the Drosophila were ready to start the climbing assay.\n\nPre-test climbing assay procedure\n\nThe tip of the climbing assay pipette was gently tapped a sufficient number of times on the bench to gather the flies together at the bottom of the apparatus, which was subsequently placed in a tube rack in an upright position at room temperature. The flies were allowed to climb for 45 seconds. There was no recording of data from this run as its purpose was to allow the flies to ‘practice’ climbing in the pipette that was held in an upright position.\n\nActual climbing assay procedure\n\nOnce the pre-test procedure had been completed, the climbing assay pipette was tapped gently on the bench (using the same number and intensity of taps as for the pre-test) and the apparatus was placed upright in the tube rack at room temperature. Drosophila were allowed to climb for 45 seconds. During the 45 seconds the number of flies to climb above the 2mL and 25mL marks were recorded. At the end of the 45 seconds, the number of flies above the 25mL mark, the number below the 2mL mark and the number in between the 2mL and 25mL mark was recorded. The whole climbing assay was repeated 2 more times to give a total of 3 readings per pipette.\n\nEnd of climbing assay procedure\n\nWhen all 3 climbing assay procedures had been performed, the Drosophila were gently tapped away from the cotton wool plug so it could be removed from the pipette without the loss of any flies. The Drosophila were then returned to fresh food vials using the dedicated plastic funnel for each group. Once the flies were back in fresh culture vials, the numbers of flies were counted and the number recorded on the lid to confirm that no flies had been lost during the assay or during the transfer to or from the pipettes. Climbing assay pipettes were checked to ensure no flies were stuck in the bottom of the pipette. The culture vials were returned to 25°C for routine fly maintenance.\n\n\nResults and Discussion\n\nDrosophila have proven to be a versatile experimental invertebrate host for use in the study of mammalian neurodegenerative diseases (Bilen & Bonini, 2005; Lu & Vogel, 2009). Several important features of Drosophila have aided this development. Firstly, Drosophila and mammals show conservation of basic components of the nervous system (Hirth & Reichert, 1999); Secondly, the genetics of Drosophila are well-defined, which allows the generation of transgenic flies with tissue-specific transgene expression. Third, the normal physiology and development of Drosophila is sufficiently well established to allow the use of behavioural assays that detect neurotoxicity in the living organism (Marsh & Thompson, 2006). Fourth, large numbers of Drosophila are readily generated in a short time and since this organism has a relatively short life span allows the rapid collection of, statistically robust data (Piper et al., 2005).\n\nIn order to develop an invertebrate-based bioassay for mammalian prion infectivity, we have generated ovine PrP transgenic Drosophila and have assessed the ability of these flies to detect ovine scrapie prions.\n\nThe Drosophila genome does not contain an orthologue of mammalian PrP and cellular expression of this protein is required for prion-induced neurotoxicity, which occurs during prion replication (Büeler et al., 1993; Mallucci et al., 2003). We exploited the successful application of PrP transgenesis to modify the susceptibility of a host for prion replication (Crozet et al., 2001; Thackray et al., 2012a; Vilotte et al., 2001) in order to explore Drosophila as a new animal model to assess mammalian prion infectivity.\n\nAlthough PrPC is primarily attached by a GPI anchor to the external side of the cell membrane, topological variants of the protein, including cytoplasmic and secreted forms, can arise during its biogenesis and metabolism (Borchelt et al., 1993; Chakrabarti et al., 2009; Hay et al., 1987; Hegde et al., 1998; Kim & Hegde, 2002; Stewart & Harris, 2003; Taylor et al., 2009). The role of these different forms of PrP in prion-mediated toxicity is not fully clarified. Accordingly, we generated Drosophila transgenic for the mature form of ovine PrP (amino acid residues 25 – 232) that was flanked by an N-terminal leader peptide and a C-terminal GPI signal peptide, which allowed expression of ovine PrP in the fly that was targeted to the plasma membrane, hereafter referred to as PrP(GPI) (Thackray et al., 2012c; Thackray et al., 2014a). In addition, we generated Drosophila transgenic for the mature form of ovine PrP that lacked the N-terminal leader peptide and C-terminal GPI signal peptide, which restricted PrP expression to the cytoplasm, hereafter referred to as PrP(cyt) (Thackray et al., 2014b). In order to generate Drosophila transgenic for these PrP variants we employed pUAST / PhiC31-mediated site-directed mutagenesis, whereby a single copy of the transgene of interest is delivered to the same landing-site in the fly genome in each respective fly line. Using this strategy, we demonstrated that different genotypes of ovine PrP protein could be successfully expressed in Drosophila. Expression of these ovine PrP variants in Drosophila had no adverse phenotypic effect upon the fly.\n\nWe subsequently tested the hypothesis that PrP transgenic Drosophila could bioassay exogenous ovine prions. To do so, Drosophila, at the larval stage, were exposed to sheep scrapie material known to contain prion infectivity as determined previously by transmission studies in mice (Thackray et al., 2008). Control inoculum consisted of known scrapie-free sheep brain homogenate. Drosophila were inoculated with scrapie-infected or scrapie-free sheep brain homogenate by addition of the material to larval feed. After hatching, flies were transferred to prion-free tubes and maintained for ≥40 days, during which time they were analysed for hallmark features of mammalian prion disease, namely the accumulation of infectious prions and evidence of a toxic phenotype.\n\nWe first investigated whether scrapie-exposed PrP transgenic Drosophila accumulated prions by measurement of prion seeding activity, a surrogate marker of PrPSc, using in vitro PMCA. Head homogenate prepared from scrapie-exposed, and control flies, was used as seed in PMCA together with brain homogenate from ovine PrP transgenic (tg338) mice as substrate. After amplification, the reaction mix was subjected to Proteinase K digest (PK) and the products analysed by western blot using an anti-PrP monoclonal antibody. Significantly, only reaction products of tubes seeded with head homogenate from scrapie-exposed PrP transgenic Drosophila showed the presence of PK-resistant PrPSc, which was good evidence for the presence of disease-associated PrP in the brains of these flies (Thackray et al., 2014a). This was supported by the presence of a potentially misfolded conformer of PrP evident by immunohistochemistry in scrapie-exposed ovine PrP transgenic Drosophila and insoluble PrP accumulation in these flies detected by conformation dependent immunoassay (Thackray et al., 2012b).\n\nWe next investigated whether bona fide infectious prions accumulated in scrapie-exposed Drosophila. This was addressed by fly-to-mouse transmission studies using ovine PrP transgenic (tg338) mice (Thackray et al., 2016). Remarkably, tg338 mice inoculated with head homogenate from scrapie-exposed PrP transgenic Drosophila developed mouse prion disease with 100% attack rate with a relatively rapid incubation time, indicative of a reasonably high level of prion infectivity in the fly head homogenate. The lack of detectable prion infectivity in head homogenate from scrapie-exposed control non-transgenic Drosophila argued against persistence of inoculum being responsible for the observed fly-to-mouse prion transmission (Thackray et al., 2016).\n\nThe presence of prion infectivity in scrapie-exposed PrP transgenic Drosophila was indicative of prion replication in these flies. Since prion-induced neurotoxicity occurs concomitantly with prion replication in mammalian hosts (Büeler et al., 1993; Mallucci et al., 2003), we investigated whether scrapie-exposed PrP transgenic Drosophila demonstrated a toxic phenotype. We assessed whether prion-exposed Drosophila showed any movement defects, since clinical signs of scrapie infection in sheep include locomotor defects, such as ataxia (Jeffrey & Gonzalez, 2007). To do so, we performed a negative geotaxis climbing assay (Nichols et al., 2012; Thackray et al., 2014a; Thackray et al., 2014b) using adult Drosophila exposed at the larval stage to ovine scrapie. After hatching, scrapie-exposed ovine PrP transgenic Drosophila showed an accelerated decline in locomotor activity. The severity of the locomotor defect increased as the flies aged, indicative of progressive illness. We also assessed whether scrapie-exposure affected the survival of PrP transgenic Drosophila since mammalian prion diseases are invariably fatal in affected individuals. Following exposure to scrapie material at the larval stage, adult PrP transgenic Drosophila showed a significantly enhanced mortality rate (Thackray et al., 2012b).\n\nCollectively, these findings demonstrated that scrapie-exposed ovine PrP transgenic Drosophila accumulated prions that were transmissible to a mammalian host. Prion accumulation in the fly was associated with a progressive toxic phenotype evident as a locomotor defect. These hallmark features of mammalian prion disease in the fly were prion-mediated and PrP dependent since the effects were not observed in PrP transgenic Drosophila exposed to normal sheep brain material and were not displayed by scrapie-exposed flies that lacked PrP expression. These observations show that PrP transgenic Drosophila can be used to bioassay mammalian prion infectivity.\n\nIn order to determine the sensitivity of the fly-based prion bioassay, ovine PrP transgenic Drosophila, at the larval stage, were exposed to a 1/10 (v/v) dilution series of scrapie-infected sheep brain homogenate. After hatching, the locomotor ability of adult prion-exposed Drosophila was assessed by a negative geotaxis climbing assay as the flies aged. We observed that the accelerated decline in locomotor ability displayed by adult PrP transgenic Drosophila diminished upon exposure to increasing dilution of scrapie-infected brain homogenate at the larval stage (Thackray et al., 2016). A statistically significant decline in locomotor ability was induced in PrP transgenic Drosophila by dilutions of scrapie-infected sheep brain homogenate in the range 10-2 - 10-10. For comparative purposes, we have used ovine PrP transgenic (tg338) mice to bioassay sheep scrapie-infected brain material. The tg338 mouse prion bioassay was able to detect sheep scrapie inoculum diluted to 10-5, with the most dilute sample detected after a time course of ≈120 days in this mouse line (Andreoletti et al., 2011). These data showed that the Drosophila-based prion bioassay is of the order 105-fold more sensitive than the tg338 mouse prion bioassay and can be completed in a significantly shorter time frame.\n\nThe high level of sensitivity shown by ovine PrP transgenic Drosophila for ovine prions suggested the fly bioassay would be able to detect the low level of prion infectivity present in the blood of prion-diseased individuals. We tested this hypothesis by inoculating ovine PrP transgenic Drosophila with plasma samples from sheep experimentally infected with scrapie (Thackray et al., 2012b). We decided to bioassay plasma since this particular blood fraction has been reported to contain low levels of prion infectivity and has proven to be difficult to assess by conventional prion bioassay (Lacroux et al., 2012; Mathiason et al., 2010). We found that PrP transgenic Drosophila developed an accelerated decline in locomotor activity that became progressively reduced after exposure to more dilute samples of scrapie-infected plasma (Thackray et al., 2016). These observations were suggestive of titration of a particulate transmissible moiety in plasma obtained from scrapie infected sheep, a distinctive feature of the infectious scrapie agent (Stamp, 1962). The sheep plasma samples were known to contain scrapie prion infectivity as they had previously been transmitted to sheep and mice (Lacroux et al., 2012).\n\nWe also observed that plasma isolated from natural scrapie-infected sheep could induce a toxic phenotype in ovine PrP transgenic flies (Thackray et al., 2016). The response to natural scrapie plasma was evident with samples collected from asymptomatic scrapie-infected sheep aged ≥6 months of age and was more pronounced after exposure to plasma obtained during the clinical phase, which commenced around 20 months of age. Importantly, we determined through fly-to-fly transmission that the toxic fly phenotype induced by pre-clinical natural scrapie plasma was transmissible (Thackray et al., 2016). These observations showed that ovine PrP transgenic Drosophila could successfully bioassay a transmissible moiety in the blood of scrapie-infected sheep, which was detectable at an early pre-clinical time point.\n\nTransfusion experiments in sheep show that whole blood from non-clinical ovine donors aged ≥3 months can be used to detect scrapie-infected animals (Lacroux et al., 2012). We consider that PrP transgenic Drosophila show a similar, if not greater, sensitivity than transfusion studies in the natural host since plasma from scrapie-affected sheep contains less prion infectivity than whole blood (Lacroux et al., 2012). Furthermore, the amount of time required to bioassay plasma in PrP transgenic Drosophila was significantly shorter than the case for transfusion studies in the natural host (McCutcheon et al., 2011).\n\n\nConclusion\n\nMany advances in prion biology have been inextricably linked to the use of experimental animals; either to model prion diseases in general or to assess prion infectivity per se. We have demonstrated that core features of mammalian prion disease, namely accumulation of disease-associated PrP and development of a transmissible toxic phenotype, can be re-capitulated in prion-exposed PrP transgenic Drosophila. Significantly, we have shown that ovine PrP transgenic Drosophila proved to be more sensitive, by several orders of magnitude, and more rapid than the ‘gold standard’ mouse bioassay for the detection of sheep scrapie prions.\n\nThese observations support the use of PrP transgenic Drosophila as a new animal system to contribute to the study of mammalian prion disease. For example, the ease of transgenesis in Drosophila will allow the development of fly lines that express different species forms of PrP, such as human, bovine and cervid PrP, in order to address important questions on the pathogenic potential of other possible zoonotic prions, such as those associated with atypical BSE and CWD. Drosophila are already used to model other protein misfolding neurodegenerative diseases. This provides considerable expertise within the scientific community to assist with the development of this tractable experimental host in an important area of animal and human health. As such, there are no significant impediments to the use of PrP transgenic Drosophila in mammalian prion disease studies.\n\nAccordingly, suitable uptake of the fly prion bioassay will be expected to have a considerable impact on the reduction and replacement, where appropriate, of more sentient hosts in the assessment of mammalian prion infectivity. In addition, the use of a Drosophila-based prion bioassay will provide a considerable refinement of the experimental protocols used to assess prion diseases. In this context, the use of Drosophila to assess mammalian prion infectivity would appear to have considerable advantages over more sentient species currently used for this purpose. Furthermore, translatability of this new invertebrate model of mammalian prion disease will be expected to provide a proof-of-concept to aid the development of new animal systems to study the prion-like properties of other neurodegenerative disease-related proteins, such as amyloid beta and tau.\n\n\nData availability\n\nAll data underlying the results presented throughout this article are available from previous publications, which have been referenced appropriately.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported in part by funds from the NC3Rs Project (Grant NC/K000462/1).\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\nAndreoletti O, Orge L, Benestad SL, et al.: Atypical/Nor98 scrapie infectivity in sheep peripheral tissues. PLoS Pathog. 2011; 7(2): e1001285. 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PubMed Abstract \| Publisher Full Text\n\nPrusiner SB: Prion biology and diseases (second edition). Vol., Cold Spring Harbor Laboratory Press, New York. 2004. Reference Source\n\nSaborio GP, Permanne B, Soto C: Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature. 2001; 411(6839): 810–3. PubMed Abstract \| Publisher Full Text\n\nStamp JT: Scrapie: a transmissible disease of sheep. Veterinary Record. 1962; 74: 357–362.\n\nStewart RS, Harris DA: Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection. J Biol Chem. 2003; 278(46): 45960–8. PubMed Abstract \| Publisher Full Text\n\nTaylor DR, Parkin ET, Cocklin SL, et al.: Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein. J Biol Chem. 2009; 284(34): 22590–600. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThackray AM, Andreoletti O, Bujdoso R: Bioassay of prion-infected blood plasma in PrP transgenic Drosophila. Biochem J. 2016; 473(23): 4399–4412. PubMed Abstract \| Publisher Full Text\n\nThackray AM, Di Y, Zhang C, et al.: Prion-induced and spontaneous formation of transmissible toxicity in PrP transgenic Drosophila. Biochem J. 2014a; 463(1): 31–40. PubMed Abstract \| Publisher Full Text\n\nThackray AM, Hopkins L, Lockey R, et al.: Propagation of ovine prions from \"poor\" transmitter scrapie isolates in ovine PrP transgenic mice. Exp Mol Pathol. 2012a; 92(1): 167–74. PubMed Abstract \| Publisher Full Text\n\nThackray AM, Hopkins L, Spiropoulos J, et al.: Molecular and transmission characteristics of primary-passaged ovine scrapie isolates in conventional and ovine PrP transgenic mice. J Virol. 2008; 82(22): 11197–207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThackray AM, Muhammad F, Zhang C, et al.: Prion-induced toxicity in PrP transgenic Drosophila. Exp Mol Pathol. 2012b; 92(2): 194–201. PubMed Abstract \| Publisher Full Text\n\nThackray AM, Muhammad F, Zhang C, et al.: Ovine PrP transgenic Drosophila show reduced locomotor activity and decreased survival. Biochem J. 2012c; 444(3): 487–95. PubMed Abstract \| Publisher Full Text\n\nThackray AM, Zhang C, Arndt T, et al.: Cytosolic PrP can participate in prion-mediated toxicity. J Virol. 2014b; 88(14): 8129–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVilotte JL, Soulier S, Essalmani R, et al.: Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp. J Virol. 2001; 75(13): 5977–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWatts JC, Prusiner SB: Mouse models for studying the formation and propagation of prions. J Biol Chem. 2014; 289(29): 19841–9. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "34033", "date": "29 May 2018", "name": "Janne M Toivonen", "expertise": [ "Reviewer Expertise Neurodegenerative diseases", "amyotrophic lateral sclerosis", "prion diseases", "Drosophila 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\nCurrently, tests of prion infectivity require an in vivo model for their propagation. These expensive and time-consuming experiments are carried out in vertebrate hosts, typically in rodents. This article offers a sound methodological overview of an alternative Drosophila bioassay developed by the authors. The Drosophila bioassay demonstrates prion-induced toxicity in the level of performance (negative geotaxis) and lifespan when subjected to primary transmission with sheep scrapie material. Bona fide acumulation of infectious prions is demonstrated by secondary transmission to PrP-expressing flies and mice using fly head homogenates from primary infection. Importantly, the described bioassay is sufficiently sensitive to detect sheep with prion disease using plasma from pre-clinical (non-symptomatic) animals. In the future, this could lead to a fast and cost-efficient blood test to diagnose human prion diseases, as well as potentially zoonotic animal diseases. Furthermore, given the ease of transgenesis in Drosophila, the described methodology could possibly be used to provide insight for potential transmissibility of other protein misfolding disorders such as Alzheimer’s and Parkinson’s diseases.\n\nAs the aim is to reduce, replace and refine, the main target audience for this methodology would probably be laboratories working with more sentinent mammalian models of prion diseases. In order to facilitate the adoption the invertebrate model for those laboratories not used to work with flies it would be useful to provide an illustrative figure describing the principles of the described bioassay, including the Gal4-system used. It would also be good mention stock numbers of used fly strains (driver lines and those used for transgenesis) to indicate that they can be easily obtained from Drosophila stock centres such as BDSC.\n\nAre a suitable application and appropriate end-users identified? Yes\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\nAre the 3Rs implications of the work described accurately? Yes\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? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "34029", "date": "12 Jun 2018", "name": "James Alibhai", "expertise": [ "Reviewer Expertise Referee suggested by the NC3Rs for their scientific expertise and experience in assessing 3Rs impact. Additional expertise: Prion disease. Rodent – Human 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 paper by Thackray et al. provides an interesting and useful overview of the development and advantages of utilising alternate methods of investigating prion infectivity, such as the Drosophila protocols outlined in this paper. The ethical implications of the paper are well discussed as are the scientific advantages of the method over the ‘gold standard’ murine studies. Overall this paper is well written, concise and a good review of the previous literature of prion-infected Drosophila research. Specific comments:\nPage 3, paragraph 4 – The authors should consider the fact that cell culture methods have been used to replicate and define human prions in human cell which have also shown capability of sub-passage between cultures1. Also recommend reference to this paper later alongside animal ‘gold standard’ comments as the sensitivity of the Drosophila model is unrivalled by human cell culture also and thus supports the Drosophila method. Page 4, preparation of drosophila head homogenate – be more explicit about how fly heads were isolated after vortex. Assume this is done manually? Are there technical issues with other parts of the fly torso impeding recognition of the fly head? If so, how do you overcome this (or does it matter)? Page 5, acclimatisation of flies in the climbing assay pipettes – this section is repetitive to the sentence above. Recommend deleting and moving information about the 30 minute incubation at 25C to sentence above. Recommend addition of simple figures that visualise the sheep à fly and fly à fly experiments with the results (or a cartoon depiction of the results).\nAre a suitable application and appropriate end-users identified? Yes\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\nAre the 3Rs implications of the work described accurately? Yes\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": [] } ]	1	https://f1000research.com/articles/7-595
https://f1000research.com/articles/7-593/v1	15 May 18	{ "type": "Review", "title": "Refinement of a mouse cardiovascular model: Development, application and dissemination", "authors": [ "Kirk A. Taylor", "Michael Emerson", "Kirk A. Taylor" ], "abstract": "European and UK legislation requires all animal procedures to be conducted with consideration to reduction, refinement and replacement. In this review, 3Rs developments are discussed in the field of platelet biology and thromboembolism. Platelet research requires the use of animal models, and mice are widely used in the field. When working in vitro, conventional light transmission techniques have been scaled down allowing reduction in animal numbers. In vivo, vascular injury models are widely used and work is ongoing to develop ex vivo approaches that use fewer animals. Thromboembolic mortality models, which inflict considerable pain and suffering, have also been used widely. A published and characterised refinement of this mortality model allows real-time monitoring of radiolabelled platelets under general anaesthesia and reduces both the severity level and the numbers of mice used in a typical experiment. This technique is more sensitive than the mortality approach and has opened up new avenues of research, which would not have been feasible by using death as an end-point. To drive uptake of real-time monitoring, a more simplistic approach has been developed involving micro-sampling and cell counting. Thromboembolic mortality models should therefore be considered obsolete due to the emergence of 3Rs models with improved scientific outcomes and that can be implemented relatively easily.", "keywords": [ "endothelium", "mortality", "mouse", "pharmacology", "platelet", "reduction refinement", "thrombosis" ], "content": "\n\n\n\nAcquisition of pharmacodynamics dose-responses increases sensitivity\n\nPlatelet function is assessed directly\n\nProcedures are conducted at a lower severity level avoiding pain and suffering.\n\nAnimal numbers are reduced by 85%\n\nSimplistic alternative micro-sampling approaches are available that require only standard laboratory equipment and minimal training\n\nInvestigating cardiovascular risk associated with exposure to airborne pollution and HIV/antiretroviral therapy\n\nBroadly within the fields of anti-thrombotic therapy and platelet physiology/pharmacology\n\nPotential for further reduction in the field by developing flow cytometric approaches for multiple analyses of diluted micro-samples\n\n\nCardiovascular diseases and platelets\n\nCardiovascular diseases including myocardial infarction (MI) and stroke are the major cause of death in Western society. Cardiovascular risk is heightened by the unavoidable risk factor of age, but is also driven by avoidable, or potentially avoidable, factors such as smoking, poor diet and airborne pollution1. Cardiovascular disease is also increasingly important to clinicians and scientists in other fields. For example, cardiovascular events are the major cause of death in people living with HIV who are virally supressed by antiretroviral therapy2.\n\nWhen investigating mechanisms of cardiovascular disease, platelets are of obvious interest. Platelets are circulating blood elements with central roles in haemostasis and thrombosis and are a major therapeutic target in the treatment of arterial thrombotic events such as MI. Aspirin and clopidogrel inhibit platelet activation and are widely prescribed in patients following MI, although their efficacy remains suboptimal and new therapeutic approaches are required.\n\nWhen activated, platelets undergo a series of signalling processes involving calcium release and the expression of activation markers, including P-selectin, on the extracellular surface. Platelets also release a range of inflammatory mediators from intracellular granules, including chemokines such as PF4 (Platelet Factor 4) and CCL5 (chemokine (C-C motif) ligand 5). Platelets therefore generate a range of signals that drive processes such as adhesion, aggregation and inflammation. Human platelets are easily isolated from blood and their function may be assessed through a range of established assays. The gold-standard functional assay remains light transmission aggregometry, which measures increases of light transmission through initially opaque platelet suspensions as platelets aggregate3.\n\n\nIn vitro and ex vivo analysis of platelet function\n\nPlatelets are anucleate and it is not possible to alter protein expression using traditional in vitro strategies, such as siRNA knockdown. Therefore, it is often necessary to employ mouse models where protein expression of the precursor cell, the megakaryocyte, can be altered. A significant reduction achievement concerning in vitro mouse platelet aggregometry has been the development of 96-well plate light transmission approaches4, which use much smaller plasma volumes than conventional cuvette approaches. By reducing the standard plasma volume from 450 to 90 μl, a reduction in mouse use of 80% is possible when switching to a 96-well format. In addition, running multiple samples in parallel in a 96-well plate, rather than consecutive cuvette analyses, can reduce sample variability and thus reduce numbers of animals required per experiment.\n\nFlow cytometry is widely used in biomedical research and with an increasing number of available antibodies and markers that permit diverse analysis in very small samples of cells and fluids, this approach can dramatically increase the number of experimental parameters per experiment. Platelet researchers routinely monitor high affinity integrin αIIbβ3, alpha and dense granule release as markers of platelet activation alongside assays to determine changes of Ca2+ signalling and aggregation5–8. Emergent flow cytometers, such as the BD Accuri C6, enable agonists to be added during sample acquisition, allowing for simultaneous kinetic evaluation of platelet activation8. A major advantage of this technology is that data can be obtained with diluted samples (i.e. 1:200), raising the possibility that micro-sampling could replace alternative blood collection techniques, such as retro-orbital sampling and cardiac puncture. Ongoing work in our laboratory is evaluating the potential of using flow cytometry to perform ratiometric analysis of agonist-evoked calcium signalling, platelet aggregation and protein studies using human samples. If successful, these techniques could be applied to mouse studies and reduce the overall number of animals required.\n\nReporting of numbers of animals used per experiment in the literature is highly variable and it is often not possible to determine whether the same animal has been used in multiple assays. Taking these limitations into account, we predict that measurement of platelet activation markers, aggregation response and calcium response to three platelet agonists using conventional assays would require 13 mice per experimental replicate. However, development of flow cytometric approaches discussed here could reduce this number by over 90%.\n\n\nNeed for animal models\n\nThe involvement of multiple cell types and tissues in the etiology of cardiovascular diseases creates a strong necessity for animal studies, and more than 63,000 animal procedures involving mice were reported in the Cardiovascular, Blood and Lymphatic field by the UK Home Office in 2016 (https://www.gov.uk/government/statistics/statistics-of-scientific-procedures-on-living-animals-great-britain-2016).\n\nWithin the field of thrombosis and platelets, vascular injury models involving endothelial damage via chemical, laser or mechanical trauma and subsequent thrombus formation are widely used9. Although the technique is conducted terminally under general anaesthesia, the number of thrombi that can be recorded per animal is limited, since, for example, both carotid arteries cannot be ligated. In addition, the high variance of the data outputs mean that relatively high numbers of animals are required to statistically power studies. Efforts are currently underway to develop an ex vivo carotid artery model involving luminal cannulation of excised artery sections: https://www.nc3rs.org.uk/reducing-animal-use-thrombosis-research-ex-vivo-injury-model. In due course, it is hoped that this model will reduce mouse use by at least 50% since both carotid arteries can be used.\n\nThe vascular endothelium releases mediators such as nitric oxide (NO) and prostacyclin, which inhibit platelet function and are central to both haemostasis and the development of platelet-driven cardiovascular events10–12. Vascular injury models have proven to be problematic when used to assess the impact of endothelial mediators in platelet-driven thrombosis. In the NO field, ablation of endothelial NO synthase (eNOS) was shown to result in no phenotype13,14, an anti-thrombotic phenotype15,16 or a pro-thrombotic phenotype17 by different groups using similar experimental approaches. Assessment of the role of endothelial mediators requires, rather than a vascular injury approach, a model in which platelets circulate freely in the context of a functional vascular endothelium, which can be manipulated pharmacologically or genetically18.\n\n\nAnimal models: Need for refinement\n\nThe need to model platelet function and thrombosis in vivo has led to the development of vascular injury models, which are conducted under general anaesthesia, but also to the use of models of thromboembolic mortality, which are conducted in conscious animals and, as the name suggests, use mortality as an end-point19. Thromboembolic mortality models allow the assessment of platelet aggregation in vivo against the backdrop of a functional vascular endothelium. Mortality models involve the intravenous injection of thrombogenic agents such as collagen or thrombin with death or hind limb paralysis as an end-point. To quote from a 2003 publication20: “Pulmonary embolism was induced in male Swiss-Webster mice by intravenous tail injection of a mixture of collagen and epinephrine. Doses were selected that resulted in death or at least 15 minutes of hind-limb paralysis in approximately 90% of control mice”. The effects of genetic modification or drug action are measured by their ability to significantly change the proportions of mice killed or paralysed. This model has been used relatively recently to assess the roles of novel intracellular signalling pathways in platelets21, endogenous compounds22, platelet receptor antagonists23, free radical scavengers24, dietary compounds25, endogenous nitric oxide10 and a novel aspirin derivative26 on thromboembolic mortality.\n\nInduction of thromboembolism in mice undoubtedly inflicts considerable pain and suffering. Following injection, the mice adopt a hunched posture, become immobile and breathing becomes laboured19. The mice remain in this state until they die or are assessed, by their inability to use their hind-limbs, to be paralysed. The rationale for conducting the procedure in conscious mice is that under anaesthetic, mortality is more difficult to induce19 (presumably shock contributes to death and blood pressure is lower under anaesthesia) and paralysis cannot easily be determined. In addition to the severe impact upon the animals, this technique requires large numbers of subjects and studies have been published involving 20 or 40 animals per experimental group10,27, so that the total number in a publication can run into the hundreds.\n\n\nRefined model development\n\nWe have developed refined models for the assessment of platelet thromboembolism using monitoring of radiolabelled platelets in anaesthetised mice28,29. Our technique avoids the use of death as an end-point and instead measures the thromboembolic response in real-time by tracking radiolabelled platelets via externally placed scintillation probes28. To achieve this, mice are terminally anaesthetised and bled by cardiac puncture, platelets are radiolabelled with a gamma emitter and then infused into a second terminally anaesthetised mouse. Thus, a model is created in which radiolabelled platelets circulate freely against the background of a fully functional vascular endothelium, and so retaining a key feature that has been used historically to justify the use of thromboembolic mortality models. The same range of thrombogenic substances that can be used to induce mortality, when given at lower doses, induce reversible and dose-dependent increases in platelet counts in a probe suspended over the pulmonary vascular bed. Thus, the whole time course of the platelet thromboembolic response may be recorded and quantified in a number of ways, including peak response and area under the curve28. We were able to validate our model with the anti-platelet drug aspirin as a means of assessing its potential clinical relevance28.\n\n\nReduction\n\nDespite the fact that real-time monitoring requires animals both for acquisition of platelets and monitoring, our approach allows not only refinement of procedures to a lower severity level, but has also reduced the numbers of animals required in a typical experiment. The extent of reduction is best exemplified when looking at work demonstrating the role of endogenous NO in inhibiting platelet activation in vivo. This was first demonstrated in mice in 1998 when administration of a NO synthase inhibitor was shown to increase thromboembolic mortality in mice10. This paper required the use of 200 mice to demonstrate the antithrombotic activity of endogenous NO and additional studies (including in vivo studies in other species) to link mortality studies with a platelet-mediated effect. A comparable study using refined real-time platelet monitoring involved only 30 mice with all procedures performed under general anaesthesia (classified as non-recovery severity level under current Home Office legislation). Thus the refined model led to an 85% reduction in mouse use in a typical experiment18.\n\n\nScientific application of refined model\n\nReal-time platelet monitoring produces dynamic and quantifiable read-outs and provides dose-dependent platelet accumulation responses. This contrasts with mortality studies which simply measure the occurrence, or lack of, an event. The refined model should therefore provide greater sensitivity since we are able to detect shifts in pharmacodynamic dose-responses, reflecting either enhanced or reduced platelet activation. We suggest that recent work in the field of cardiovascular risk in the context of airborne pollution demonstrates this30,31. It has been known for many years that exposure to pollution leads to increased cardiovascular morbidity and mortality and, in particular, exposure to particulate pollution increases myocardial infarction, a platelet-driven cardiovascular event32. Due to their physicochemical properties, combustion-derived nanoparticles such as diesel exhaust particles (DEP) have been strongly implicated in driving cardiovascular risk33. DEP have been shown to induce inflammation34 and due to their nanoparticulate nature are hypothesised to translocate across the pulmonary epithelial barrier into the blood35, which would bring them into direct contact with circulating platelets. Having shown that DEP can interact physically with platelets and induce their aggregation in in vitro human studies30, it was necessary to proceed to in vivo studies to translate this finding to a more relevant whole organism setting. It was shown that both introduction of DEP to circulating blood to mimic translocation30 and tracheal instillation of DEP to mimic inhalation31 resulted in a significantly enhanced platelet thromboembolic response at doses of DEP that reflected human exposure levels. These studies contributed to the now more widely accepted thinking that DEP exposure may enhance cardiovascular risk in the human population and provided a potential mechanism. We suggest that this type of work, which highlights relatively subtle events that require sensitive models, would not have been possible in mortality models, certainly not at doses of DEP that were relevant to human exposure levels.\n\n\nDissemination and uptake\n\n3Rs benefits of animals arise not from their development, which creates potential, but from their subsequent employment in studies where non-refined models would have been used in the absence of refined alternatives. Examples from our own group have been discussed above and there are other outcomes in the fields of nanotoxicology36, nutritional biochemistry37 and calcium signalling38. We have also collaborated with other groups in the fields of cyclooxygenase pharmacology12, integrin linked kinase8 and sulforophane (an isothiocyanate with potential antithrombotic activity; unpublished study, authors: Gillespie, Holloway, Becker, Rauzi, Vital, Shreveport, Taylor, Stokes, Emerson and Gavins).\n\nThe ultimate aim of 3Rs research is broad uptake of emerging 3Rs technologies by the scientific community and a consequential reduction in the use of non-refined procedures. Quantification of continued use of thromboembolic mortality models is difficult since their use is often obscured through lack of inclusion in abstracts and use of vague terminology. PubMed searches of [thromboembolism + mouse] and [thrombosis + mortality + mouse] and [platelet + mortality + mouse], followed by a manual search to identify publications revealed more than 50 papers where thromboembolic mortality was used in 2013. A review of these papers found that all used mortality and/or paralysis as end-points, the duration of the experiments varied from 5 minutes to 96 hours, and experimental groups varied from 10 to 30 mice, with 10 to 360 mice used in total in each paper. Similar numbers of papers appear in the 5 years preceding 2013.\n\nMore recently, in 201639, we sampled 9 peer review articles using models of thromboembolic mortality and found between 3 and 40 animals per experimental group, with some studies not reporting animal use, the duration of the period in which animals were observed following induction of thromboembolism varied from 5 min to 5 days. Anaesthesia of any sort was only used in one study. Unfortunately, thromboembolic mortality models are therefore still used despite the availability of an alternative refined model. Potential reasons for continued use of thromboembolic mortality models are: Lack of awareness of alternatives, animal welfare not a primary consideration, inability to work with radioisotopes, lack of expertise and preference for collaboration rather than establishment of new technologies\n\nIncreasing awareness of the 3Rs and refined models is an ongoing endeavour and we continue to work collaboratively. In addition, we set out to develop refined methods to study platelet thromboembolism in vivo that could be more widely adopted since they could be conducted with minimal training, non-specialised equipment and at low cost. We have now developed a model that allows thromboembolism to be assessed by measuring the fall in circulating platelets that occurs during thromboembolism in blood microsamples39. Microsamples are taken from the tail vein of anaesthetised mice and repeated sampling allows for counts to be measured before and during the thromboembolic response in an individual animal. This technique allows thromboembolism to be assessed without the need for specialised equipment and without radioisotopes.\n\n\nConclusions\n\nIn conclusion, platelet function can now be assessed in vitro and in vivo using 3Rs approaches that reduce the severity level and also the numbers of animals used in procedures. Thromboembolic mortality approaches should now be considered obsolete, since even where the gold-standard real-time monitoring technique cannot be employed, more simplistic assays can be used without the need for radioactive material or complex procedures.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by research grants from the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) (grant numbers: G0600382/1; G0900732/1; NC/M000079/1).\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\nMills NL, Donaldson K, Hadoke PW, et al.: Adverse cardiovascular effects of air pollution. Nat Clin Pract Cardiovasc Med. 2009; 6(1): 36–44. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEmerson M: Refinement, reduction and replacement approaches to in vivo cardiovascular research. Br J Pharmacol. 2010; 161(4): 749–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiMinno G, Silver MJ: Mouse antithrombotic assay: a simple method for the evaluation of antithrombotic agents in vivo. Potentiation of antithrombotic activity by ethyl alcohol. J Pharmacol Exp Ther. 1983; 225(1): 57–60. PubMed Abstract\n\nMatys T, Kucharewicz I, Pawlak R, et al.: Nitric oxide-dependent antiplatelet action of AT1-receptor antagonists in a pulmonary thromboembolism in mice. J Cardiovasc Pharmacol. 2003; 42(6): 710–3. PubMed Abstract \| Publisher Full Text\n\nJin YR, Hwang KA, Cho MR, et al.: Antiplatelet and antithrombotic activities of CP201, a newly synthesized 1,4-naphthoquinone derivative. Vascul Pharmacol. 2004; 41(1): 35–41. 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PubMed Abstract \| Publisher Full Text\n\nMomi S, Emerson M, Paul W, et al.: Prevention of pulmonary thromboembolism by NCX 4016, a nitric oxide-releasing aspirin. Eur J Pharmacol. 2000; 397(1): 177–85. PubMed Abstract \| Publisher Full Text\n\nPark J, Lee B, Choi H, et al.: Antithrombosis activity of protocatechuic and shikimic acids from functional plant Pinus densiflora Sieb. et Zucc needles. J Nat Med. 2016; 70(3): 492–501. PubMed Abstract \| Publisher Full Text\n\nTymvios C, Jones S, Moore C, et al.: Real-time measurement of non-lethal platelet thromboembolic responses in the anaesthetized mouse. Thromb Haemost. 2008; 99(2): 435–440. PubMed Abstract \| Publisher Full Text\n\nHolbrook L, Moore C, Sanz-Rosa D, et al.: A NOD/SCID mouse model for the assessment of human platelet aggregation in vivo. J Thromb Haemost. 2012; 10(3): 490–2. PubMed Abstract \| Publisher Full Text\n\nSolomon A, Smyth E, Mitha N, et al.: Induction of platelet aggregation after a direct physical interaction with diesel exhaust particles. J Thromb Haemost. 2013; 11(2): 325–34. PubMed Abstract \| Publisher Full Text\n\nSmyth E, Solomon A, Birrell MA, et al.: Influence of inflammation and nitric oxide upon platelet aggregation following deposition of diesel exhaust particles in the airways. Br J Pharmacol. 2017; 174(13): 2130–2139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeters A, Dockery DW, Muller JE, et al.: Increased particulate air pollution and the triggering of myocardial infarction. Circulation. 2001; 103(23): 2810–5. PubMed Abstract \| Publisher Full Text\n\nDonaldson K, Tran L, Jimenez LA, et al.: Combustion-derived nanoparticles: a review of their toxicology following inhalation exposure. Part Fibre Toxicol. 2005; 2: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBéruBé K, Balharry D, Sexton K, et al.: Combustion-derived nanoparticles: mechanisms of pulmonary toxicity. Clin Exp Pharmacol Physiol. 2007; 34(10): 1044–50. PubMed Abstract \| Publisher Full Text\n\nKreyling WG, Semmler-Behnke M, Seitz J, et al.: Size dependence of the translocation of inhaled iridium and carbon nanoparticle aggregates from the lung of rats to the blood and secondary target organs. Inhal Toxicol. 2009; 21 Suppl 1: 55–60. PubMed Abstract \| Publisher Full Text\n\nSmyth E, Solomon A, Vydyanath A, et al.: Induction and enhancement of platelet aggregation in vitro and in vivo by model polystyrene nanoparticles. Nanotoxicology. 2015; 9(3): 356–64. PubMed Abstract \| Publisher Full Text\n\nApostoli GL, Solomon A, Smallwood MJ, et al.: Role of inorganic nitrate and nitrite in driving nitric oxide-cGMP-mediated inhibition of platelet aggregation in vitro and in vivo. J Thromb Haemost. 2014; 12(11): 1880–9. PubMed Abstract \| Publisher Full Text\n\nJones S, Solomon A, Sanz-Rosa D, et al.: The plasma membrane calcium ATPase modulates calcium homeostasis, intracellular signaling events and function in platelets. J Thromb Haemost. 2010; 8(12): 2766–2774. PubMed Abstract \| Publisher Full Text\n\nRauzi F, Smyth E, Emerson M: Refinement of Mouse Protocols for the Study of Platelet Thromboembolic Responses In Vivo. Thromb Haemost. 2017; 117(12): 2283–2290. PubMed Abstract \| Publisher Full Text" }	[ { "id": "34028", "date": "22 May 2018", "name": "Sarah Jones", "expertise": [ "Reviewer Expertise Platelets", "endothelial cells", "thrombosis" ], "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 availability of appropriate platelet function assays and thrombosis models is crucial to study mechanisms, which regulate the initiation and development of platelet rich thrombi, responsible for myocardial infarction and stroke. The study of specific proteins through altering expression levels in vitro is not possible in platelets since they do not have a nucleus. The use of genetically modified mice has therefore been crucial in furthering our understanding of platelet biology over the last two decades. European and UK legislations require that all animal procedures, are given suitable consideration to the 3Rs; reduction, refinement and replacement. In this review, Taylor and Emerson highlight some of the key developments in the platelet field, promoting the 3Rs principles, and discuss recent application of these developments. Critically, the review emphasises that impact on the 3Rs, can only be achieved through uptake of alternative technologies by the scientific community, which remains a significant challenge.\n\nThe review gives a brief but comprehensive summary of recent techniques, which have had a significant impact on reducing animal numbers used in ex vivo platelet studies. These techniques allow for smaller blood volumes to yield meaningful functional data, reducing the number of animals sacrificed. Further on going research optimising flow cytometry techniques by the Emerson group, may ultimately lead to the acquisition of platelet function and signalling data, in blood samples obtained by micro-sampling. This could replace the need for blood collection methods such as cardiac puncture, which are performed under terminal anaesthesia.\n\nA brief mention is given to the existing vascular damage models of thrombosis. The authors should be cautious in this section, to be specific with the model that they are referring to and avoid over-generalization. While it is true that arterial thrombosis, mediated by ferric chloride (FeCl3 ) and measured by vascular occlusion can only asses one thrombi per animal and data is variable, this is not the case for laser injury, analysed by intravital microscopy. In this model, in excess of 20 thrombi can be analysed per animal and as few as 3 animals used in each experimental group1,2.\n\nThe review highlights the importance of endothelial derived platelet inhibitors in regulating thrombosis and haemostasis, and using nitric oxide as an example, indicates that vascular injury models have yielded contradicting results. While I am in agreement with this, I do not think that the approaches in the cited studies can be referred to as similar. Differences in animal age, the mode and extent of vascular damage, and the type of vessel studied likely reflects the different results reported. Even studies using FeCl3 to injure the carotid artery vary significantly when FeCl3 is applied at different concentrations or for different lengths of time3.\nAs part of the discussion around measuring endothelial contributions to thrombosis, the authors state ‘Assessment of the role of endothelial mediators requires, rather than a vascular injury approach, a model in which platelets circulate freely in the context of a functional vascular endothelium, which can be manipulated pharmacologically or genetically.’ While this may be correct in a specific context, the appropriate model to investigate the role of endothelial derived mediators depends very much upon the research question. To investigate, the role of endothelial derived inhibitors on platelet aggregation in vivo, a model where platelets are circulating freely with a functional endothelium is appropriate. If however the contribution of the endothelium to arterial thrombosis or antithrombotic efficacy is being investigated, models, which encapsulate platelet adhesion, aggregation and thrombus stability in arterial conditions, following vascular damage, are more appropriate. It is also important to note that endothelial dysfunction is common in many diseases, which predispose to thrombosis, however currently there are no in vitro or in vivo models available to investigate the impact of this on thrombus generation.\n\nThe review gives an excellent summary of the thromboembolism model developed by Emerson and colleagues, with well supported statistics of the significant impact on 3Rs that it has had so far. There is also good reference to application of the model in a variety of studies. There is good critique pertaining to limited uptake of the model by the scientific community, and potential reasons for this are discussed. The Emerson group have developed a further model, which is cost-effective, requires minimal training and non-specialised equipment, to try and address some of these issues and overcome the challenge of increased uptake. In summary, this review highlights important work carried out by the Emerson group, supported by the National Centre for the Replacement, Refinement and Reduction of Animals in Research, which has successfully developed, validated and applied refined real-time thromboembolisms models to replace the severe and archaic thromboembolism model that uses mortality or hind limb paralysis as an end-point.\n\nDoes the review provide an accurate account of the 3Rs landscape in that field / across fields? Yes\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": "34026", "date": "23 May 2018", "name": "David J. Grieve", "expertise": [ "Reviewer Expertise Referee suggested by the NC3Rs for their scientific expertise and experience in assessing 3Rs impact. Additional expertise: cardiovascular remodelling", "heart failure", "oxidative stress", "endothelial proenitor cells", "diabetes", "experimental models", "3Rs." ], "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 article describing development of an improved experimental model of thromboembolism with clear potential to both replace and refine existing models. Whilst the manuscript is generally well written and presented, I do think that it could be significantly improved by revising structure and increasing 3Rs emphasis. Specific comments below.\n\nThe title of the article does not accurately reflect its content. It is too general and should be specifically focussed on thromboembolism with regard to development of an improved experimental model. The stated scientific benefits in the research highlights box could also be made more specifically and understandable on a stand-alone basis.\n\nThe article would benefit from inclusion of one of two figures to complement the text and make the article more engaging. For example, a summary of current experimental models used in platelet research with approximation of numbers of animals used would be helpful and it would also seem appropriate to include a schematic representation of the new model highlighting potential benefits.\n\nNeed for animals models: the flow of this section could be improved. I do not think that the final paragraph really fits as currently written. More specific detail should also be provided in relation to the provided NC3Rs link e.g. research group etc.\n\nWith specific regard to thromboembolism models, some discussion in relation to where these procedures are currently performed (e.g. are they permitted in the UK?) and comparison of scientific end-points between the new and existing models would be informative in order to highlight relevance and applicability.\n\nDissemination and uptake: this section is quite weak at present and could be improved by including discussion of e.g. current barriers to uptake and potential approaches to encourage wider implementation. At present, discussion is largely limited to the authors own collaborators and in line with NC3Rs policy more needs to be done (which I am sure that it is) to promote wide uptake of new experimental models across the field.\n\nAbstract – specify light transmission aggregometry techniques.\nDoes the review provide an accurate account of the 3Rs landscape in that field / across fields? Yes\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": "34024", "date": "25 May 2018", "name": "Alan G.S. Harper", "expertise": [ "Reviewer Expertise Platelet physiology", "calcium signalling", "vascular tissue engineering" ], "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\nMice are widely used in the platelet research field - this is largely due to this species’ accessibility to genetic modification and intravital microscopy. However due to their small blood volume and size and the variable responses to surgical treatments, these experiments often require the use of significant number of mice to reproduce the experiments to get statistically significant data. Therefore there is a key need to identify 3Rs strategies to help platelet researchers minimise the harm done to mice used in this field.\n\nIn this report, the authors provide an interesting and insightful review into the 3R’s approaches that have been applied to both ex vivo and in vivo studies of platelet function in mice. Taylor and Emerson provide excellent examples of how 3Rs approaches are being used and developed in their laboratory to help refine and reduce the use of mice in platelet research. Of the different approaches, the refinement of the thromboembolic model provides a particularly valuable case study into how 3Rs approaches can not only improve animal welfare but also improve the quality of the scientific data obtained.\n\nWhilst the authors discuss barriers to the dissemination of their technique, it would have been interesting to have seen more detailed description of these barriers, and a consideration of potential ways they could be overcome to further increase the 3Rs impact of this work. For example, a more detailed assessment of the training, financial and infrastructure needs required to adopt this technique and how this lead to the development of the cell counting model, would have provided an interesting insight into how to improve the impact of 3Rs interventions.\n\nThe discussion of the uses of the thromboembolic and vascular injury models identifies some key differences between these models, however the review seems to more consider the relative merits of these two techniques as approaches to studying thrombosis generally, rather than perhaps more clearly describe their complementary roles in examining thrombosis and haemostasis – with the vascular injury models better replicating normal haemostatic reactions in the absence of a complete endothelial lining rather than true arterial thrombosis, whilst the thromboembolic model better reflects the processes of venous thromboembolism where coagulation is artificially triggered inside the intact venous system. However the key trigger of the acute cardiovascular events mentioned by the authors in their introduction is atherosclerotic plaque rupture. The review therefore would be benefit from a brief mention of animal models of plaque rupture to give a more complete picture of the field. Current murine atherosclerosis models are hampered by the lack of spontaneous plaque rupture with models using requiring a mechanical trigger to elicit the full pathological response1, therefore future 3Rs work may also be needed on common non-mouse models in rabbits, pigs and non-human primates2.\nFor ex vivo studies, the authors provide some interesting case studies on how adoption of improved technology can pave the way to reduce blood volumes required to perform these studies, and as such reduce the number of mice utilised. The authors show that these have the potential to have a major impact in reducing the number of animals. However this section does not appear to fit with the title of the review – therefore the review is brief and omits some promising 3Rs ex vivo approaches. The most important omission is of studies aiming to replace the use of mice in platelet research by attempting to recreate the physical and chemical environments of the circulation ex vivo using microfluidic and tissue engineering techniques3. These are beginning to provide a viable alternative to current animal models, and should also be considered as a potential future target of 3Rs interventions in platelet research.\nDoes the review provide an accurate account of the 3Rs landscape in that field / across fields? Partly\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/7-593
https://f1000research.com/articles/6-2120/v1	11 Dec 17	{ "type": "Software Tool Article", "title": "Simulation and visualization of multiple KEGG pathways using BioNSi", "authors": [ "Adva Yeheskel", "Adam Reiter", "Metsada Pasmanik-Chor", "Amir Rubinstein", "Adva Yeheskel", "Adam Reiter", "Metsada Pasmanik-Chor" ], "abstract": "Motivation: Many biologists are discouraged from using network simulation tools because these require manual, often tedious network construction. This situation calls for building new tools or extending existing ones with the ability to import biological pathways previously deposited in databases and analyze them, in order to produce novel biological insights at the pathway level. Results: We have extended a network simulation tool (BioNSi), which now allows merging of multiple pathways from the KEGG pathway database into a single, coherent network, and visualizing its properties. Furthermore, the enhanced tool enables loading experimental expression data into the network and simulating its dynamics under various biological conditions or perturbations. As a proof of concept, we tested two sets of published experimental data, one related to inflammatory bowel disease condition and the other to breast cancer treatment. We predict some of the major observations obtained following these laboratory experiments, and provide new insights that may shed additional light on these results. Tool requirements: Cytoscape 3.x, JAVA 8 Availability: The tool is freely available at http://bionsi.wix.com/bionsi, where a complete user guide and a step-by-step manual can also be found.", "keywords": [ "biological network", "simulation", "gene expression", "KEGG", "Cytoscape", "BioNSi" ], "content": "Introduction\n\nModeling and simulation of biological regulatory networks is becoming an integral part of biological research nowadays1. Such networks consist of nodes, representing, e.g., proteins, RNA, genes, nutrients, signals, etc. Edges represent the interactions between nodes, such as activation, inhibition, phosphorylation, etc. Simulation tools allow the analysis and visualization of the dynamics of these networks. Biological data, such as protein interactions and gene or protein expression are gathered and provide the required input for such tools. Indeed, in recent years many bioinformatics tools aiming at the simulation of biological networks have been published. The purpose of such tools is to elucidate the relationships between genes, proteins, pathways, or other biological entities involved, to shed new light on the experimental results, and to suggest possible directions for future research.\n\nSimulation tools come in various flavors. Some focus on transcriptional regulatory networks, others on signal propagation or metabolic pathways. There are tools that require programing skills, while others provide a complete graphical user interface (GUI). The underlying mathematical models differ a lot too. Perhaps the most fundamental distinction is between continuous, Boolean and discrete models. Continuous network models typically apply differential equations, using real (as opposed to discrete) numbers to represent the system’s variables (e.g., 2, while in Boolean network models variables may assume only one of two values, namely 0 (“OFF”, or inactive) and 1 (“ON”, or active) (e.g., 3. Between the two \"extremes\" are discrete models, in which values are taken from a range of integers (e.g., 0, 1, 2, …9). This is often a good compromise between the expressiveness of the model and its simplicity, and it is especially useful when only partial or imprecise biological knowledge of these systems is available4.\n\nTable 1 provides a summary of several network modeling and simulation tools properties. These tools are relatively new ones (most of them published within the last four years). In addition, we focused on biologist-friendly tools that require very little computational background and in particular no script writing skills. The last tool in Table 1 is BioNSi - Biological Network Simulator5, whose extensions are the focus of this paper, as will be described later.\n\nA representative list of seven network simulation tools was obtained from OmicsTools. A summary of some important features is shown. V: denotes that a specific feature exists in the tool. Web/App: a web tool or downloadable software. Nodes levels: Boolean (B)/discrete (D)/continuous (C). Asynchronous update: an option for updating specific nodes before others. (e.g., by defining delay or priorities). Batch simulation mode: running a collection of simulation with various parameters to reveal emergent network properties. Edges weights: weights represent strength of interaction. Network generation: textual (T)/graphical (G). A star (*) represents features implemented in the extended BioNSi tool (but not in the original version).\n\nIn Table 1 we compare the following features:\n\n– Web/App: whether the tool is web-based (web) or a stand-alone software or application (app)\n\n– Nodes' levels: the granularity of the model variables, that is, whether nodes levels are Boolean (B), discrete (D) or continuous (C) variables\n\n– Asynchronous update: whether the node level transition rules can be applied asynchronously, rather than simultaneously (for example, by specifying delays on edges, or defining priorities on edges)\n\n– Batch simulation mode: whether the tool supports a batch mode of simulation, in which multiple initial conditions are explored simultaneously and global (emergent) properties such as the system steady states (attractors) are identified\n\n– Edge weights: whether the edges in the network have weights to represent strength (amplitude) of interaction, in contrast to merely +1 (activation) and -1 (inhibition)\n\n– Network generation: which format is supported for network generation - graphical (G), in which the network is drawn on the canvas, or textual (T), in which the user generates a table of nodes and edges\n\n– Merge pathways: whether the tool supports merging of multiple pathways of interest into a single network.\n\n– Upload expression data: whether expression data from e.g., laboratory experiments can be loaded into the network automatically.\n\nNetwork simulation tools typically require manual network construction based on biological data. For networks beyond a very small scale of a few nodes, this process becomes tedious, time consuming, and error prone. Furthermore, conducting numerous simulations of a network under different biological conditions may require a tiresome and monotonous process of modifying network parameters for each set of conditions. This contributes to the discouragement of many biologists from integrating network simulation tools in their daily laboratory routine. This situation is extremely unfortunate, since a lot of network data based on lab experiments already exists in various common databases. Therefore, the integration of network simulation tools with biological data previously deposited into those databased provides novel opportunities for more comprehensive studies of these networks. Initiatives aiming at such integration may support biological understanding of previous laboratory results, and direct researchers to design new experiments.\n\nTo this end, we have integrated a recently published tool, BioNSi (Biological Network Simulator5 with the KEGG pathway database information12. BioNSi was originally used for visualizing biological networks and simulating their time course behavior. For the sake of self-containment of this paper, Box 1 describes BioNSi at a high level. A full user guide and a step by step manual can be found on the tool's website (http://bionsi.wix.com/bionsi).\n\n\n\nBioNSi is a biological network simulation tool5. It allows constructing a regulatory network and simulating its behavior. The network consists of nodes (interactors) and edges (interactions). For example, nodes may represent genes, RNA or transcription factors, while edges represent interactions such as transcription, translation, phosphorylation, etc.\n\nFor each node, the user manually defines an initial expression level, termed initial state, an integer typically taken from the range between 0 and 9 (0 – no activity, 9 – high activity). Time in the model is also discrete, represented as clock \"tics\" or time steps (t = 0, 1, 2,…). Nodes' states are updated in each time step by a transition rule (see below).\n\nEach edge is assigned a weight which reflects the strength of the interaction between 2 nodes, and is either a positive integer (activation, translation, etc.) or a negative one (repression, inhibition, degradation, etc.). Degradation usually appears as a self-negative edge. The effect of node i on node j is the product of node i's state and the weight of the edge i→j. Thus, the strength of this effect is proportional to both the weight of the edge and the relative level of activity of the source node. The \"net\" effect on a node is the sum of effects of all nodes on it. Figure A shows a \"toy\" network with 4 nodes (A, B, C, signal; colored rectangles), whose initial states are shown below their names. Edges are labeled with their weights and colored accordingly (green = activation; red = inhibition).\n\nFigure A: a \"toy\" network in BioNSi\n\nFigure B: graphical presentation of simulation results\n\nA simulation starts with a given configuration of initial states, and consists of the repeated application of a transition rule, simultaneously to all nodes: the state of a node will increase, decrease, or remain unchanged when the \"net\" effect on it is, respectively, positive, negative or 0. The simulation ends when either a steady state is reached (two consecutive identical configurations of nodes' states), or a loop of configurations is detected.\n\nThe tool presents the course of simulation graphically (see Figure B). The network exhibits an infinite loop in which the states of nodes A, B and C repeatedly go up and down, while the signal node is constant at state 1. BioNSi also enables a batch mode, which is used to gather statistics on a set of simulations, to study global properties of the network (e.g., the distribution of steady states, average time to get to each steady state, etc.).\n\nBioNSi supports various additional features, which are described in the manual on the website, such as delay on edges, blocking edges, nodes that represent external signals with a pre-defined expression pattern whose behavior is pre-set by the user, etc.\n\nOur new developments enable merging of multiple KEGG pathways into a single coherent network under the BioNSi framework, and uploading experimental expression data to conduct a simulation of the network. The initial expression levels of network nodes (genes, proteins, etc.) may be obtained either from in-house experiments or from various expression databases (e.g.,, GEO13 or ArrayExpress14. This allows easy simulation of the resulting network under different experimental conditions (expression values). For example, one can simulate the effect of inhibitors, activators, mutations, abnormal cell conditions or the effect of a new drug on the network's behavior, suggesting further experimental exploration. BioNSi is a Cytoscape application15. Cytoscape is an open source bioinformatics software platform, which hosts various applications for visualization and analysis of networks. The benefit of this is that the BioNSi simulation results can be further extended with additional analyses based on other applications. The typical workflow with the new version of BioNSi is presented in Figure 1.\n\nGene expression data and selected KEGG pathway data-files are uploaded to BioNSi’s Cytoscape Application and analyzed. The resulting network can be modified and tuned. Simulation is performed for gene expression data resulting from various conditions, possibly with addition of artificial perturbations. Differences in the results of simulation under various conditions provide novel biological insights.\n\nWe first describe the details of our new extensions to BioNSi. Then, as a proof of concept, we demonstrate the advantages and usability of our tool by presenting the simulation and analysis of previously published experimental data from two sets of experiments: (1) the characterization of inflammatory bowel disease (IBD) conditions, and (2) treatment of MCF7 breast cancer cells with heregulin (HRG). These two test cases may provide insights into how our tool can be used to elucidate existing biological data, raise new hypotheses and suggest new lab experiments to test them.\n\n\nMethods\n\nCytoscape is an open source software platform for complex network analyses and visualization15. It enables \"plugging in\" in the form of dedicated software called applications (Apps), which are developed by users. BioNSi5 is one such application that provides a user friendly tool for simulating the dynamics of biological regulatory networks. It is implemented under Cytoscape version 3. Installation is easy and explained in the tool's website at http://bionsi.wix.com/bionsi. We have extended BioNSi’s capabilities with two key features presented in the next sections.\n\nImporting and merging multiple KEGG pathways into BioNSi. The original version of BioNSi allowed creating networks “de novo”, by adding nodes and edges manually, according to the biological knowledge of the researcher. This may be a tedious task, when the networks are beyond a very small size. The new feature allows easily importing multiple pathway files that were exported from the KEGG database. The pathways are simply downloaded from KEGG as xml files, and merged into a single, integrated network under BioNSi. The user guide on BioNSi's website explains the details for this operation. Briefly, nodes contained in several pathways are fused into a single node, with all their incoming and outgoing edges. The user can specify the desired weights for each KEGG type of edge, such as activation, inhibition, phosphorylation, etc., or rely on the default values provided. As biological materials are routinely being degraded, \"self-inhibition\" loops are automatically added to each node. Subsequently, the resulting network can be \"tuned\" by adding or removing specific nodes and edges, or by changing specific parameters (e.g., the weight of a specific edge). Thus, constructing large-scale integrative networks from known pathways is done with little effort, and enables inspecting biological pathways at a broader biological context.\n\nImporting nodes’ initial states. The second new feature allows the initial nodes' states obtained either from in-house experiments or from various expression databases to be loaded from a simple text file (a two-column comma separated values (csv) text file, in which the left column contains nodes' names and the right one contains the desired initial levels). Such a file can be easily generated from most existing gene expression databases, using e.g., Microsoft Excel. Since existing expression data sources may have different scales (e.g., logarithmic vs. linear), nodes' initial states are read from the input file and normalized to the range 0–9 for consistency and convenience. In addition, we extended BioNSi with a graphical representation of the nodes' levels: nodes' background colors indicate their initial states (higher levels are darker gray). Once a simulation ends, nodes colors are automatically updated to reflect their final state, providing a visual image of the incline or decline in nodes' expression. This new feature allows easy comparison between the network's behavior under different experimental conditions, by loading alternative sets of initial states and conducting a new simulation each time.\n\nSimulation. In the two test cases described in the Use Cases section, we conducted simulations for 100 time-steps. All simulations reached a steady state within this number of steps. In order to compare between simulation results, we had to define when a node is considered to exhibit different behavior in the two simulations. We used a simple Euclidian distance measure between the two simulation trajectories of each node. Furthermore, we filtered out nodes whose difference in the two simulations is merely a result of a different initial state. We did that by considering only nodes for which the difference between the simulations, at some time step, was larger than the difference in the initial state. In other words, the gap between the node's states in the two compared simulations should increase at some point beyond the initial gap, in order for it to be considered differential between the simulations. An implementation of this distance function can be found as a Python script (diff.py) that can be downloaded from the tool's website.\n\n\nUse cases\n\nIBD are a series of chronic inflammations of the intestine. Pouch surgery is a useful treatment of IBD patients, but pouch inflammation is a very common outcome16. Crohn’s-like disease of the pouch (CLDP) is the most severe inflammation condition. Activation of NFKB was identified as one of the key regulators inducing IBD inflammation by promoting pro-inflammatory cytokines17,18. Parthenolide, a strong NFKB inhibitor, has recently been demonstrated to be a promising therapeutic agent in IBD inflammation, promoting apoptosis of cancer cells19. We aim at extending the original study analyzing differentially expressed genes and their functional enrichment, to suggest new targets for treatment.\n\nExpression data source. Affymetrix GeneChip (Human Gene 1.0 ST arrays) was used for gene expression analysis of normal and CLDP donors, as previously described16.\n\nPathway data. As reported in 16, 74 genes were found to be up-regulated in CLDP vs normal expression (pFDR<0.05 and fold-change difference=5). WebGestalt KEGG pathway enrichment20 revealed enriched 16 pathways (pBonf<0.05), of which the top 5 were selected for analysis (Table 2). In addition, IBD pathway was added, although not specifically enriched in the analysis (total of 6 pathways).\n\nTop human KEGG pathways were obtained and enrichment p-values presented. Links to KEGG xml file download are provided.\n\nNetworkAnalyzer out-degree analysis. Network analysis was performed using NetworkAnalyzer tool, which is another built-in Cytoscape application21. We used it in order to visualize the most out-connected (notably high out-degree) genes in the network. The most outstanding such node is NFKB1 (19 outgoing edges, see Table 3). Therefore, an additional NFKB signaling pathway (hsa04064) was added from KEGG. Altogether, 7 KEGG pathways were analyzed, composed of 379 nodes (mostly genes).\n\nCytoscape’s NetworkAnalyzer tool was used to present out-degree score. NFKB1 has an outstanding out-degree score.\n\nIn the resulting merged network (Figure 2), in addition to genes, compounds and complexes were also detected (presented as triangles and hexagons, respectively). These were automatically assigned expression value 0 as no information could be found for them in the expression data.\n\nWe show only 241 out of 379 genes, which belong to the main connected subnetwork resulting from 7 KEGG pathways (excluding subnetworks of 8 nodes or less). We used Cytoscape’s hierarchical layout, in which nodes with no outgoing edges appear at the top. Gene’s fill color is according to control expression values (grayscale), except nodes that are different between the simulation of CLDP and control expression, which are marked in red, and NFKB1 which is colored turquoise.\n\nEdge weights. We used edge weights as following: inhibition -1, activation +2. As biological materials are being degraded over time, self-inhibition edges (weight -1) were automatically added for each node in the network. Accordingly, dephosphorylation (-p) edges are set to -1. Activation edges, including phosphorylation (+p) were set to +2, to compensate for the degradation edges automatically added to all nodes.\n\nSimulation of different experimental conditions. We aimed at comparing network simulations of normal and CLDP donor expression values. One of the new features in BioNSi enables uploading a new configuration of initial states into an existing network. All simulations were conducted for 100 steps and reached steady state within this number of steps. In order to compare between simulation results, we had to define when a node is considered to exhibit different behavior in the two simulations. We used a simple Euclidian distance measure between the two simulation trajectories of each node. Furthermore, we filtered out nodes whose difference in the two simulations is merely a result of different initial states. We did that by considering only nodes for which the difference between the simulations, at some point in the simulation, was larger than the difference in the initial state. In other words, the gap between the node's states in the two compared simulations should increase at some point beyond the initial gap. An implementation of this distance function can be found as a Python script on the tool's website (diff.py).\n\nResults. We aim at comparing between two simulations: 1) Control gene expression; 2) CLDP gene expression. The network consists of 379 nodes. The comparison revealed 49 nodes that exhibited different simulation trajectories (genes, compounds and complexes; 12.9% of all nodes in network). 87.8% of these (43 nodes) were downstream in the network, namely, had no outgoing edges (except for self-loops that all nodes have). To visualize this we used Cytoscape’s hierarchical layout, as shown in Figure 2. The nodes that differed between the two simulations are colored red. The hierarchical layout positions these nodes at the top of the network. NFKB1 gene (highlighted in turquoise) is highly connected within the network, but not to any of the selected (red) genes.\n\nA large fraction of the network’s genes is not directly connected to the main network, but forms small independent sub-networks (138 nodes with 8 nodes or less). This is a result of selecting only 7 KEGG pathways for the analysis. Naturally, the analysis is highly dependent on the KEGG pathways selected. Disconnected sub-networks may be connected to the main network following the inclusion of additional, less enriched KEGG pathways. However, these subnetworks follow the same pattern seen in the main network, and all of the nodes differing in the 2 simulations are at the top of the hierarchy (Supplement 1; http://www.cs.tau.ac.il/~amirr/files/supp/testcase1_files.zip). For clarity, Figure 2 shows only the main connected subnetwork (241 nodes; 138 nodes were removed from Figure 2, all harboring connected subnetworks with 8 or less nodes, presented in Supplement 1).\n\nIn terms of biological function, most of the 49 nodes that exhibit differences between the simulations are of genes that are either immune system receptors (receptors for chemokines, cytokines, interferons, interleukins, PDGF, growth factors (MET) etc.), or various kinases (BTK, MAPK14, MAP3K7, IKBKB, FLT3 and FLT4). Many therapeutic agents are offered based on such molecules22. Several cytokines (that may be targeted by JAK inhibition), were found within the BioNSi resulting nodes that exhibited different simulation trajectories: IL6ST, IL6R and OSMR (gp130 family member), IL10RB (IL10 members), IL2RB, IL7R (γc family) and CSF1R (βc family), and IFNAR1, IFNAR2 (interferon related genes)23. This analysis may suggest interesting new targets for drug response.\n\nThe purpose of the following example is to demonstrate the effect of HRG treatment on MCF7 breast cancer cells, based on published GEO experimental results (GSE6462;24,25. The simulation results suggest that BioNSi is capable of recapitulating the major changes in the network after drug treatment, as observed by in vitro experiment.\n\nExpression data source. MCF7 breast cancer cells were treated with a growth hormone, HRG, at four different doses and different time course (GSE6462). We used expression data after maximal treatment of 10 nM HRG for 90 minutes. In the experiment, the mRNA expression levels were measured for control (GSM148517) and HRG treated (GSM148572) cells (using Affymetrix Human Genome U133A 2.0 Arrays.\n\nPathway data. KEGG-xml files for three selected human pathways were exported from the KEGG database12: ERBB signaling pathway (hsa04012); MAPK signaling pathway (hsa04010); EGFR cytosine kinase inhibitor resistance pathway (hsa01521). These three representative pathways were reported to be crucial for the HRG treatment response24. The merged network based on these three KEGG pathways consisted of 190 nodes.\n\nSetting network parameters and post-import modification. We used edge weights as follows: inhibition -1, activation +2. As biological materials are being degraded over time, self-inhibition edges (weight -1) were automatically added for each node in the network. Accordingly, dephosphorylation (-p) edges are set to -1. Activation edges (including phosphorylation; +p) were set to +2, to compensate for the degradation edges automatically added to all nodes. A node representing HRG treatment was added manually (diamond shape), either with initial state 0 (without drug), or with initial state 9 (active drug). The HRG node was directly connected to its known target nodes, ErbB3 and ErbB4, in addition to their homodimers nodes, with strong inhibition edges (weight -10 each). Protein homodimers (resulting from KEGG database) were given expression values similar to their monomer molecules (from the microarray mRNA expression results). Heterodimers were given initial state 0, as we have no data concerning their actual expression.\n\nSimulations. A useful application of the BioNSi tool is to compare between the simulation results under different nodes' initial states (reflecting different biological conditions). As illustrated in Figure 3, we compared simulations of control (A) vs HRG treatment (B) gene expression, in addition to simulation of artificial HRG effect on control gene expression (C).\n\nExpression data from GSE6462 experiment was obtained for Control MCF7 breast cancer cells, without (A) or with (B) HRG treatment. (C) BioNSi simulation of artificial HRG effect performed on control expression.\n\nIn order to simulate artificial HRG treatment effects on the network, we used control expression values as initial states, and the HRG node at initial state 9. Network’s simulation under these conditions resulted in a complete and rapid decline of ErbB3, ErbB4 and their homodimer expression values.\n\nResults. We aimed at comparing between two simulation sets, based on the same network created: (1) Simulation that compares between HRG gene expression vs. control gene expression. The comparison between these simulations revealed 132 different genes (69.5% of all genes in the network (190)) whose expression pattern differs between the simulations of HRG vs Control. (2) Simulation of HRG artificial treatment effect on the same control gene expression, vs. control expression. This comparison revealed 33 genes (17% of the total 190 in network) that were found to be different. Figure 4 shows a Venn diagram that demonstrates these findings. 28 genes are common to both analyses. Artificial HRG treatment results (28 genes) represent ~21% of the experimental results (132 genes). 5 nodes were found to be uniquely changed by the prediction analysis, including the obviously changed (ERBB3, ERBB4 and ERBB4-ERBB4, which are directly inhibited by HRG simulation prediction, in addition to STAT5A).\n\n28 genes were common to both analyses (pink). 5 genes were unique to simulation (orange), and 104 genes were unique to experimental analysis (blue).\n\nInteresting observations concerning the resulting genes can be observed in the BioNSi screenshot of the network in Figure 5. All the 33 genes (framed pink and orange), resulting from the artificial HRG treatment simulation analysis compared to control, are highly connected, especially downstream of the network. This suggests an interesting signal path of the HRG drug response, which may offer new targets for treatment. We note that the PI3K/AKT/mTOR and STAT pathway genes are predominantly changed following HRG artificial treatment prediction, pathways that may affect cell proliferation and apoptosis, as recently suggested24,25.\n\nA network of 190 genes consisting of 3 KEGG pathways (described in the text), using control expression values is shown. Gene frames color code: common genes to the two analyses (pink), unique to prediction of artificial HRG treatment vs control (orange), unique to simulation resulting from HRG treatment vs control expression analysis (blue) or unchanged (black). The network shows genes with nodes fill color grayscale according to expression values and edge colors according to type of interaction (activation–green; inhibition-red). For convenience, genes or proteins are represented as rectangles, compounds as triangles and complexes (dimers) are hexagons. HRG treatment is presented as orange-framed diamond.\n\n\nConclusions\n\nThe bio-technological revolution we are witnessing in recent decades enables generating unprecedented amounts of biological data of various types. This includes expression levels of RNA and proteins, and the interactions between them and other substances, such as nutrients, hormones, external stimuli etc. While these data are accessible to researchers, their analyses at the system level are less so. Extracting specific and local information is easy, but performing large scale network analyses requires computational approaches, such as simulation tools. However, existing network simulation tools are often meant for manual construction by the researcher of the network under study, and therefore remain inefficient for studying networks beyond a small scale. This is extremely unfortunate in an era when computational approaches to study biological systems are proliferating. Network simulations are one such important computational approach, which has proven efficient in providing new insights, shedding light on existing results, and suggesting new directions for exploration, both in the lab and on the computer. In order for simulation tools to become an integral part of the researchers' lab routine, tools that enable simulation of large scale network data are called for. Such tools must avoid a tedious network construction process, and allow a high level representation of networks to be generated from existing data semi-automatically with little effort and no computational skills.\n\nIn this paper we suggest an extension to an existing network simulation tool, BioNSi, that allows researchers to import multiple pathway data from the KEGG database into a single network representation. The tool then allows for the study of the network via simulations, under different biological conditions. We believe such an approach will foster the use of computational tools in the lab by researchers with little or no computational background.\n\n\nSoftware and data availability\n\nTool requirements: Cytoscape 3.x, JAVA 8\n\nTool download: http://bionsi.wix.com/bionsi (a full user guide and a step by step manual can be found at the tool's website)\n\nTool source code: http://doi.org/10.5281/zenodo.106535226\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.106535226\n\nLicense: CC BY 4.0\n\nData source for test case 1: https://www.ncbi.nlm.nih.gov/pubmed/24108111\n\nData source for test case 2: ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE6nnn/GSE6462/matrix/GSE6462_series_matrix.txt.gz", "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 Varda Wexler from the Multimedia Unit in the Faculty of Life Sciences, Tel Aviv University, for creating the BioNSi logo, and Benny Chor from the School of Computer Science at Tel Aviv University for useful comments during manuscript preparation.\n\n\nSupplementary material\n\nTest case 1 files: http://www.cs.tau.ac.il/~amirr/files/supp/testcase1_files.zip\n\nTest case 2 files: http://www.cs.tau.ac.il/~amirr/files/supp/testcase2_files.zip\n\n\nReferences\n\nBarabási AL, Oltvai ZN: Network biology: understanding the cell’s functional organization. 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PubMed Abstract \| Publisher Full Text\n\nSchivo S, Scholma J, van der Vet PE, et al.: Modelling with ANIMO: between fuzzy logic and differential equations. BMC Syst Biol. 2016; 10(1): 56, [cited 2017 Jan 26]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBock M, Scharp T, Talnikar C, et al.: BooleSim: an interactive Boolean network simulator. Bioinformatics. 2014; 30(1): 131–2, [cited 2017 Jan 26]. PubMed Abstract \| Publisher Full Text\n\nKanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000; 28(1): 27–30, [cited 2017 Jan 26]. 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): D991–5, [cited 2017 Jul 19]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKolesnikov N, Hastings E, Keays M, et al.: ArrayExpress update--simplifying data submissions. Nucleic Acids Res. 2015; 43(Database issue): D1113–6, [cited 2017 Jul 19]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSu G, Morris JH, Demchak B, et al.: Biological network exploration with Cytoscape 3. Curr Protoc Bioinformatics. 2014; 47: 8.13.1–24, [cited 2017 Jul 19]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBen-Shachar S, Yanai H, Baram L, et al.: Gene expression profiles of ileal inflammatory bowel disease correlate with disease phenotype and advance understanding of its immunopathogenesis. Inflamm Bowel Dis. 2013; 19(12): 2509–21, [cited 2017 Jul 19]. PubMed Abstract \| Publisher Full Text\n\nAtreya R, Neurath MF: New therapeutic strategies for treatment of inflammatory bowel disease. Mucosal Immunol. 2008; 1(3): 175–82, [cited 2017 Oct 17]. PubMed Abstract \| Publisher Full Text\n\nAhmed S, Dewan MZ, Xu R: Nuclear factor-kappaB in inflammatory bowel disease and colorectal cancer. Am J Dig Dis. 2014; 1(2): 84–96, [cited 2017 Oct 18]. Reference Source\n\nKim Y, Kim TK, Kim Y, et al.: Principal network analysis: identification of subnetworks representing major dynamics using gene expression data. Bioinformatics. 2011; 27(3): 391–8, [cited 2017 Jan 26]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang J, Duncan D, Shi Z, et al.: WEB-based GEne SeT AnaLysis Toolkit (WebGestalt): update 2013. Nucleic Acids Res. 2013; 41(Web Server issue): W77–83, [cited 2017 Jan 26]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDoncheva NT, Assenov Y, Domingues FS, et al.: Topological analysis and interactive visualization of biological networks and protein structures. Nat Protoc. 2012; 7(4): 670–85, [cited 2017 Oct 17]. PubMed Abstract \| Publisher Full Text\n\nNeurath M: Current and emerging therapeutic targets for IBD. Nat Rev Gastroenterol Hepatol. 2017; 14(11): 688, [cited 2017 Oct 26]. PubMed Abstract \| Publisher Full Text\n\nDe Vries LCS, Wildenberg ME, De Jonge WJ, et al.: The Future of Janus Kinase Inhibitors in Inflammatory Bowel Disease. J Crohns Colitis. 2017; 11(7): 885–93, [cited 2017 Oct 26]. PubMed Abstract \| Publisher Full Text\n\nNagashima T, Shimodaira H, Ide K, et al.: Quantitative transcriptional control of ErbB receptor signaling undergoes graded to biphasic response for cell differentiation. J Biol Chem. 2007; 282(6): 4045–56, [cited 2016 Nov 15]. PubMed Abstract \| Publisher Full Text\n\nRubinstein A, Bracha N, Rudner L, et al.: BioNSi: A Discrete Biological Network Simulator Tool. J Proteome Res. 2016; 15(8): 2871–80, [cited 2017 Jan 26]. PubMed Abstract \| Publisher Full Text\n\nRubinstein A: BioNSi - Biological Network Simulation Tool (Version 1.2). Zenodo. 2017. Data Source" }	[ { "id": "29573", "date": "05 Feb 2018", "name": "Aurélien Naldi", "expertise": [ "Reviewer Expertise Qualitative modeling of regulatory networks" ], "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 presents an extension of the BioNSi modeling software with two aims: (i) kickstart model construction by integrating KEGG pathways and (ii) use experimental expression data as initial states for the simulation. These aims make sense: facilitating the initial construction and evaluation of dynamical models is an important challenge in systems biology. However, I feel that details on the inner working and discussion of the limitations of this tool are missing from the paper. Some related work should also be taken into account. In particular Wrzodek et al.1 and Büchel et al.2 which generate qualitative models in SBML qual format3 from KEGG pathways. My main concerns are detailed in the following text, none of them would be blocking taken separately, but my overall interpretation is that this work requires deep changes to become convincing.\nI installed the BioNSi tool in Cytoscape 3.4. The documentation states that building a network from KEGG pathways requires the selection of a folder containing one or several KEGG xml files and a CSV file with expression data. I tried to provide a folder containing two KEGG pathways and an empty CSV file, but it did not work (error message: \"number of expression files is not 1\"). I had the same problem with Cytoscape 3.6.\nKEGG pathways often \"merge\" multiple biological entities in a single node, the paper does not say how this case is handled, the documentation only states that ellipsis dots are removed from the name. Does it mean that these groups are preserved? How does this affect merging multiple KEGG pathways? It can be a concern as the same entity could be involved in different groupings in different KEGG pathways.\nMore generally, it would make sense to separate the transformation of KEGG pathways into discrete models (taking into account previous work1,2), and the merging of multiple discrete models, which is a more general problem, and could be used to integrate several existing models with new models generated from pathway maps.\nThe import of initial states from experimental data, and in particular the discretization step deserve further discussion. For a generic tool, I suspect that it uses a simple method where the full range of values for all genes is scaled to 0-9, defining 9 evenly-spaced thresholds. Is it the case or are the thresholds based on the range of each gene separately? As mapping experimental data on the model is one of the two core aims of this paper, it should at least review the alternatives, give proper details on how this is handled and state that alternative mapping can be integrated as a preprocessing step.\nMapping entities in the model to entities in the experimental data is not discussed either, does the tool convert KEGG identifiers to official gene names, does it require the user to do the mapping in advance, does it provide a GUI to fine-tune the mapping afterwards?\nThe paper does not state what is the objective of using an experimentally measured values as initial state. The experiments presented here seem to represent \"steady states\" of the biological system, the simulation leads to a steady state which can be compared to the experimentally measured one. Differences between the experimental and computed states can be of two types: (i) inconsistency showing that the model needs further improvement, and (ii) effect that is not measured in the experiment: for example change of phosphorylation which is not measured by expression data. As this is the very core of the paper, a discussion on the possible biological meaning of the results seems required.\nThe BioNSi tool, which this work extends, supports simulations of qualitative (discrete) models. Its capabilities are summarized in this paper, and it is briefly compared to other tools for qualitative modelling in Table 1. A minor typo in this table replaced BooleSim with BoolSim (there is also a tool called BoolSim in this field: it is the command line tool handling the Boolean part of SQUAD). The paper describes BioNSi (and some other tools) as doing synchronous and asynchronous simulations, but misuses the term \"asynchronous\". Here asynchronous refers to synchronous with delays or priorities (i.e. all transitions do not happen at the same speed, but are still using the same clock), however it usually means independent trajectories, leading to non-deterministic behaviour. Using the classical asynchronous updating of qualitative models, a state can have several separate successors, each corresponding to the update of a single component. This allows to study systems where the relative speeds of transitions is not known, but introduces numerous concurrent trajectories, making the analysis much harder. GINsim is the only tool in the provided list implementing asynchronous updating, it also supports priorities, but not delays. BoolNet4, PyBoolNet5, and Boolsim6 support asynchronous updating. To handle this complexity, some tools such as BooleanNet7 or MaBoSS8 perform random walk in the asynchronous dynamics.\nIn BioNSi, inhibitions have negative weights, while activations have positive weights. These weights are multiplied by the level of their source nodes, the resulting weighted sum is then used to define the evolution of the target element. While this allows to quickly define a model, I am concerned by the lack of flexibility: some inhibitors only block the effect of one of the activators, some regulators have a cooperative effect, some act independently. I agree that the simple weight system used in BioNSi is convenient when these fine-grained mechanisms are unknown: it provides a good first model. However, the KEGG pathways mostly describe processes which have been deeply studied, and for which such an approximation could be avoided. There is some value in a simple system based on weights, especially as it fits better for integration into cytoscape, but building more precise functions through the use of intermediate nodes representing cooperative effects should at least be discussed as a common refinement.\nSome minor points:\nMissing closing of 2 parenthesis on page 2, another on page 3 Page 6: \"138 nodes with 8 nodes or less\" is confusing. I suspect it means 138 nodes within sub-networks of 8 nodes or less? The first paragraph on page 6 (\"simulation of different experimental conditions\") repeats the last paragraph of the methods.\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? 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? Partly", "responses": [ { "c_id": "3633", "date": "14 May 2018", "name": "Amir Rubinstein", "role": "Author Response", "response": "Dear Aurélien,We greatly appreciate the detailed report. Below please find our point-by-point reply. Changes were made to the manuscript in its 2nd version. To ease reading of the responses below, we copy-pasted the comments in the report as well. --------------------------------------- Some related work should also be taken into account. In particular Wrzodek et al.1and Büchel et al.2 which generate qualitative models in SBML qual format3 from KEGG pathways. My main concerns are detailed in the following text, none of them would be blocking taken separately, but my overall interpretation is that this work requires deep changes to become convincing. Regarding the additional resources, they were added to the Introduction. Our focus is at integrating a Cytoscape application (BioNSi) with KEGG, exploiting this convenient simulation environment. I installed the BioNSi tool in Cytoscape 3.4. The documentation states that building a network from KEGG pathways requires the selection of a folder containing one or several KEGG xml files and a CSV file with expression data. I tried to provide a folder containing two KEGG pathways and an empty CSV file, but it did not work (error message: \"number of expression files is not 1\"). I had the same problem with Cytoscape 3.6. In the current version of the tool the csv expression file should not be empty. This is obviously something to fix in the next version. In the meantime, we provide such a file with arbitrary initial levels of 9. The file can be downloaded from the website and from http://www.cs.tau.ac.il/~amirr/files/expression.csv. Both Cytoscape 3.4 and 3.6 should work fine. KEGG pathways often \"merge\" multiple biological entities in a single node, the paper does not say how this case is handled, the documentation only states that ellipsis dots are removed from the name. Does it mean that these groups are preserved? How does this affect merging multiple KEGG pathways? It can be a concern as the same entity could be involved in different groupings in different KEGG pathways. BioNSi preserves all the alternative names for a KEGG entity. When merging pathways, even if two nodes have different alternative names, they will be fused into a single node. Furthermore, names of entities in the expression level file are matched against any of the alternative names of a node in the network. This information was emphasized in the new version of the user manual and paper. More generally, it would make sense to separate the transformation of KEGG pathways into discrete models (taking into account previous work1,2), and the merging of multiple discrete models, which is a more general problem, and could be used to integrate several existing models with new models generated from pathway maps. Such separation of functionality indeed makes a lot of sense. It may be implemented as part of a later version of the tool. The import of initial states from experimental data, and in particular the discretization step deserve further discussion. For a generic tool, I suspect that it uses a simple method where the full range of values for all genes is scaled to 0-9, defining 9 evenly-spaced thresholds. Is it the case or are the thresholds based on the range of each gene separately? As mapping experimental data on the model is one of the two core aims of this paper, it should at least review the alternatives, give proper details on how this is handled and state that alternative mapping can be integrated as a preprocessing step. The initial levels are scaled to 0-9 for all the nodes. The following was added to the paper: \"Note that the initial states are scales to 0-9 for the whole network based on the maximal expression level in the network, rather than for the range of each gene separately. Since the scaling depends of the maximal expression level of a node in the network, an extreme outlier may restrict the levels of other nodes to a very limited range. Therefore, the use of logarithmic expression data is recommended, and when the raw data in not logarithmic a simple transformation is required as a preprocessing step.\" Mapping entities in the model to entities in the experimental data is not discussed either, does the tool convert KEGG identifiers to official gene names, does it require the user to do the mapping in advance, does it provide a GUI to fine-tune the mapping afterwards? We use KEGG xml files which include the complete array of gene\\protein names available, including official gene symbol, HGNC id, KEGG id and others. In BioNSi, we use all gene symbols for each entry and therefore, identifiers are not being lost. For the graphical presentation, the first identifier is being used. The paper does not state what is the objective of using an experimentally measured values as initial state. The experiments presented here seem to represent \"steady states\" of the biological system, the simulation leads to a steady state which can be compared to the experimentally measured one. Differences between the experimental and computed states can be of two types: (i) inconsistency showing that the model needs further improvement, and (ii) effect that is not measured in the experiment: for example change of phosphorylation which is not measured by expression data. As this is the very core of the paper, a discussion on the possible biological meaning of the results seems required. We added such a discussion to the Methods section (Simulation): \"We explored two use-cases as a proof of concept, as described in the next section. In both use cases, KEGG pathways were integrated to form a BioNSi network, and gene expression analysis was used as initial nodes' states. In both use cases, the states of nodes changed throughout the simulation process, terminating in a steady state. This change in states may reflect one of two situations: (i) the expression data itself does not represent a biological steady state, and therefore the changes in-silico reflect changes in-vivo, and (ii) data is missing from the network, either structural (missing regulators or connections between existing nodes) or quantitative (weights on edges, initial states, etc.). In the latter case the conclusion is that the model needs further improvement. \" The BioNSi tool, which this work extends, supports simulations of qualitative (discrete) models. Its capabilities are summarized in this paper, and it is briefly compared to other tools for qualitative modelling in Table 1. A minor typo in this table replaced BooleSim with BoolSim (there is also a tool called BoolSim in this field: it is the command line tool handling the Boolean part of SQUAD). Fixed. The paper describes BioNSi (and some other tools) as doing synchronous and asynchronous simulations, but misuses the term \"asynchronous\". Here asynchronous refers to synchronous with delays or priorities (i.e. all transitions do not happen at the same speed, but are still using the same clock), however it usually means independent trajectories, leading to non-deterministic behaviour. Using the classical asynchronous updating of qualitative models, a state can have several separate successors, each corresponding to the update of a single component. This allows to study systems where the relative speeds of transitions is not known, but introduces numerous concurrent trajectories, making the analysis much harder. GINsim is the only tool in the provided list implementing asynchronous updating, it also supports priorities, but not delays. BoolNet4, PyBoolNet5, and Boolsim6 support asynchronous updating. To handle this complexity, some tools such as BooleanNet7 or MaBoSS8 perform random walk in the asynchronous dynamics. We changed \"asynchronous\" to \"non-simultaneous\", and added this column to Table 1. In BioNSi, inhibitions have negative weights, while activations have positive weights. These weights are multiplied by the level of their source nodes, the resulting weighted sum is then used to define the evolution of the target element. While this allows to quickly define a model, I am concerned by the lack of flexibility: some inhibitors only block the effect of one of the activators, some regulators have a cooperative effect, some act independently. I agree that the simple weight system used in BioNSi is convenient when these fine-grained mechanisms are unknown: it provides a good first model. However, the KEGG pathways mostly describe processes which have been deeply studied, and for which such an approximation could be avoided. There is some value in a simple system based on weights, especially as it fits better for integration into cytoscape, but building more precise functions through the use of intermediate nodes representing cooperative effects should at least be discussed as a common refinement. BioNSi (already in the original version) does support additional regulation effects in the form of blocking and enabling edges and external signal nodes. These are only mentioned without details in the end of Box 1. We added a brief description of these in box 1. Some minor points:Missing closing of 2 parenthesis on page 2, another on page 3 We cannot find these missing parenthesis, we would appreciate a clarification. Page 6: \"138 nodes with 8 nodes or less\" is confusing. I suspect it means 138 nodes within sub-networks of 8 nodes or less? Fixed. The first paragraph on page 6 (\"simulation of different experimental conditions\") repeats the last paragraph of the methods. We deleted the redundant paragraph from the results." } ] }, { "id": "29782", "date": "21 Feb 2018", "name": "Giovanni Scardoni", "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 tried to simulate the toy example from the BioNSi website, following the step by step guide. It doesn't work. Tried some other examples (cellcyclesB, Testcase1_network) and no results. Maybe there is a bug (I am using Cytoscape 3.6.0) or there is something I'm missing. In the second case the step by step guide should be improved.\nThis doesn't allow me to provide a complete evaluation of the tool and the paper that seems interesting and well written. I will be happy to reconsider my review once such problems are solved.\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? Partly", "responses": [ { "c_id": "3484", "date": "14 May 2018", "name": "Amir Rubinstein", "role": "Author Response", "response": "Dear Giovanni, Thank you for the prompt feedback.After carefully reviewing the data again, we found a bug in opening the toy.cys network on some combinations of Cytoscape, Java and operating systems. We are working on fixing it. In the meantime, we double checked and the csv format should work properly on all versions. Therefore, we temporarily removed the cys files from the examples tab, leaving only the csv files. In addition, we slightly changed the step-by-step pdf manual to match these changes.We hope this will allow a complete evaluation of the new version of BioNsi. Thank you,Amir" } ] } ]	1	https://f1000research.com/articles/6-2120
https://f1000research.com/articles/7-229/v1	26 Feb 18	{ "type": "Research Article", "title": "Birds of primary and secondary forest and shrub habitats in the peat swamp of Berbak National Park, Sumatra", "authors": [ "Kevin F.A. Darras", "Dedi Rahman", "Waluyo Sugito", "Yeni Mulyani", "Dewi Prawiradilaga", "Agus Rozali", "Irfan Fitriawan", "Teja Tscharntke", "Dedi Rahman", "Waluyo Sugito", "Yeni Mulyani", "Dewi Prawiradilaga", "Agus Rozali", "Irfan Fitriawan", "Teja Tscharntke" ], "abstract": "Background: Tropical lowland rainforests are threatened by deforestation and degradation worldwide. Relatively little research has investigated the degradation of the forests of South-east Asia and its impact on biodiversity, and even less research has focused on the important peat swamp forests of Indonesia, which experienced major losses through severe fires in 2015. Methods: We acoustically sampled the avifauna of the Berbak National Park in 2013 in 12 sites split in three habitats: primary swamp forest, secondary swamp forest, and shrub swamp, respectively representing non-degraded, previously selectively logged, and burned habitats. We analysed the species richness, abundance, vocalisation activity, and community composition across acoustic counts, sites, feeding guilds and IUCN Red List categories. We also analysed community-weighted means of body mass, wing length, and distribution area. Results: The avifauna in the three habitats was remarkably similar in richness, abundance and vocalisation activity, and communities mainly differed due to a lower prevalence of understory insectivores (Old-World Babblers, Timaliidae) in shrub swamp. However primary forest retained twice as many conservation-worthy species as shrub swamp, which harboured heavier, probably more mobile species, with larger distributions than those of forest habitats. Conclusions: The National Park overall harboured higher bird abundances than nearby lowland rainforests. Protecting the remaining peat swamp forest in this little-known National Park should be a high conservation priority in the light of the current threats coming from wildlife trade, illegal logging, land use conversion, and man-made fires.", "keywords": [ "primary forest", "secondary forest", "shrub swamp", "swamp forest", "community ecology", "forest disturbance", "forest fires", "selective logging" ], "content": "Introduction\n\nWe are losing tropical forests and their associated biodiversity worldwide to deforestation, and this loss is irreplaceable1. Forest loss also occurs because of degradation: selective logging affects large areas2, which in turn become more susceptible to other disturbances like fire3. Most studies focus on the Amazon region, but forests in Southeast Asia also face great threats4. Forest losses due to fire have recently gained more attention in Indonesia5,6. Indonesia has the highest deforestation rate of all countries7, and even protected forests suffer losses8. The effectiveness of protecting forests in Indonesia has been questioned for Kalimantan9, but for Sumatra, modest progress has been made, especially as large-scale logging has slowed down10.\n\nThe impacts of forest loss and degradation on biodiversity are better known in the Amazon region, for instance for birds11. In Southeast Asia, most primary forest bird species still occur in previously logged forests12–14. Globally, bird feeding guilds respond differently to disturbance15, but forest understory insectivores were identified as the most sensitive16. The severity of the disturbance and its impact on soils largely determine the duration of recovery, while the surrounding landscape acts as a source of biota17.\n\nWe lack studies investigating the impact of disturbances - from logging or fire - on bird communities in peat swamp forests, despite their crucial importance for biodiversity, flood control, carbon stocks, potential greenhouse gas emissions, and their high vulnerability to drainage and fires18,19. Few studies have focused on the potential benefit of disturbances for overall landscape biodiversity apart from theoretical and modelling approaches20,21.\n\nIn this paper we describe the bird communities in a tropical peat swamp on Sumatra, Indonesia. Bird surveys of the Berbak area date far back22,23. We sampled birds in three habitats defined according to Keddy (2010)24: primary swamp forest, secondary swamp forest resulting from selective logging, and shrub swamp resulting from forest fire. We compare species richness and bird communities between these habitats both taxonomically (species richness, abundance) and functionally (vocalisation activity, body mass, wing length, distribution area). We ask the question whether disturbances increase the overall diversity of the landscape. We discuss the implications of our results for maintaining the overall bird diversity of the peat swamp and for bird conservation in Berbak.\n\n\nMethods\n\nWe surveyed birds in Berbak National Park, a peat swamp situated in the province of Jambi, on the east coast of the island of Sumatra in Indonesia (Figure 1). Berbak National Park is a Ramsar site25 and an Important Bird Area26. A severe drought in 1997 facilitated forest fires which were aided by human disturbance due to natural rubber collection (Dyera costulata). The burned areas subsequently developed into shrub swamp. Tree stumps also reveal that illegal selective logging affected several areas of the park, resulting in secondary forest habitats.\n\nWe chose 12 sites for counting birds, divided in four sites for each of three habitat types of the peat swamp area, representing different past disturbance levels: primary forest (no disturbance), secondary forest (selective logging), and shrub swamp (fire).\n\nField surveys.\n\nWe recorded audible sound in all 12 sites. We used autonomous sound recorders (SM2+ recorders with SMX-II microphones, Wildlife Acoustics Inc.) and did not carry out visual surveys, since acoustic recording constitutes a valid survey method for assessing bird richness27, especially for cryptic birds in tropical forests. From February to November 2013, we sampled all three habitats at one site each month for 48 hours, starting at midnight. Before the recording started, we counted and measured the diameter at breast height (DBH) of all trees with a circumference above 20 cm (diameter at breast height of ~6.4 cm) inside an area of 14 × 14 m delimited by spanning 10 m coloured ropes from the central tree where the sound recorder was attached to all cardinal directions. We also recorded whether the site was flooded or not at the time of the sound recorder installation.\n\nWe uploaded 20 minutes recordings after sunrise for each plot in each month (24 recordings in total) to our online platform (http://soundefforts.uni-goettingen.de/). The provenance of the recordings was hidden and all birds within the sound recordings were identified by author IF. The distance of bird vocalisations was estimated by ear to the meter, based on their loudness in the sound recording compared to the ambient sound level and knowledge of the source sound level of each species. The start and end time of each vocalization was recorded to compute the duration of the bird vocalisations. We complemented our data set with species-specific information about the feeding guild28,29, body mass data28,30, wing length data31, the IUCN Red List threat status32 and distribution area33 to analyse conservation and functional aspects of bird community differences.\n\nThe data were analysed in R 3.4.3 and graphs were generated with the package ggplot234. We excluded detections that were not identified to species, as well as detections above 50 m to compare sites at a common detection radius35. We computed species richness, bird vocalizing activity in minutes, and bird abundance per acoustic count (i.e. recording). Bird species richness was further computed at the site and habitat level. Bird abundance was counted as the sum of the maximum number of individuals vocalising simultaneously in each species. We calculated community-weighted means (or community functional parameter36, hereafter CWM) for body mass, wing length, and distribution area for each count. Due to microphone failure, the second recording of one of the forest plots had only one audible audio channel and was therefore removed from the count-level analysis.\n\nAt the count level, the species richness and abundance between habitats was modelled using generalised linear mixed effects models of the poisson family (lme4 package37), with site as random variable, and we checked that the models were not over-dispersed. Similarly, vocalizing activity was analysed with a linear mixed effects model. At the site level, we used generalised linear models of the poisson family to model alpha and beta bird species richness, which we calculated using the additive partitioning approach38. For all models, we used tree number, tree basal area, and habitat type as predictors. We generated all possible predictor combinations and compared the models using Akaike's Information Criterion for small sample sizes (herafter AICc, MuMIn package, dredge function) to choose the best model (with the lowest AICc).\n\nWe visualized the composition of the bird communities in non-metric multidimensional scaling graphs generated with the package vegan39, and tested the significance of the habitat in structuring these communities with an ADONIS test40. We also plotted the abundance of birds within different families in each of the habitats along with their conservation status. To investigate whether the combined, different habitats lead to higher species richness than one primary forest area of similar size, we calculated the rarefied richness based on the entire bird community, rarefied to 4 sampling sites, to compare it to the number of species found in the 4 forest sites.\n\n\nResults\n\nWe detected 379 birds overall, belonging to 90 species (Table S1). Among those, 26 individuals were not identified to species and 2 were detected above 50 m, resulting in a working dataset of 351 detections. The three habitats differed considerably based on their vegetation structure and the distribution of their DBH values (Figure S1 and Figure S2).\n\nSpecies richness and abundance at the count level, and mean alpha and beta species richness at the site level were similar between the habitats (Figure 2 and Figure 3).\n\nTotal (gamma) richness for each habitat type, split by alpha and beta richness components. Error bars show the 83% confidence intervals for the mean alpha and beta richness values. Overlapping bars indicate that the means are not significantly different.\n\nThese variables were best explained by null models. Gamma species richness per habitat was as follows: primary forest: 50 species, secondary forest: 52 species, bush swamp: 50 species. Bird richness and abundance between habitats, split into different functional groups and IUCN red list threat categories, were similar between habitats (Figure S2).\n\nBird vocalisation activity at the count level was similar among habitats (Figure 3) and best explained by a null model. CWMs of body mass, wing length and distribution area however were increasing along the disturbance gradient and the CWMs in shrub swamp were significantly higher than in primary forest. Wing length CWM was also higher in shrub swamp than in secondary forest (Figure 3).\n\nBird species richness, abundance, vocalizing activity, community-weighted mean (CWM) of body mass, wing length, and distribution area33 per count in three different habitats of the peat swamp of the Berbak national park. Mean values are represented with red dots and their 83% confidence intervals are indicated with error bars. Means are significantly different when their confidence intervals do not overlap, and significant differences are indicated with asterisks.\n\nBird communities differed greatly between primary forest and bush swamp, with secondary forest being an intermediate habitat having overlap with both of the latter (Figure S3). The ADONIS test revealed that habitat structured bird community composition with marginal significance (P=0.053). The difference in the bird communities arose mainly from the higher abundance of Timaliidae (Old World babblers) in the forest habitats. Pycnonotidae (Bulbuls) and Alcedinidae (Kingfishers) were more prevalent in shrub swamp, while Nectarinidae were more frequent in the forest habitats (Figure 4).\n\nAbundances are split by habitat and shown for different IUCN Red List statuses.\n\n\nDiscussion\n\nWe have shown that all three habitats inside the Berbak peat swamp forest have similar abundance, vocalizing activity and species richness. The habitat, tree number or basal area were not related to these measures. Even the richness and abundance of the bird feeding guilds and abundances inside families were similar between habitats. However, we detected differences in the bird community composition, notably a decrease in understory insectivores (Old World Babblers, Timaliidae) which was compensated by the appearance of other species in shrub swamp.\n\nThe Berbak peat swamp forest is threatened from many sides. It is difficult to access as only waterways (irrigation canals and rivers) lead into it, and large parts are temporarily flooded due to tides. However, wild bird extraction for the caged bird market, illegal logging, land-use conversion at its margins, and natural rubber (jelutung) collection are all currently happening41. These human activities increase the risk of fires, especially during dry spells caused by the warm phases of the El Niño Southern Oscillation, which led to the especially severe fires of 1994, 1997, and 2015 (after our survey).\n\nPrimary forest sites had high conservation value because we found 16 species of conservation concern (\"near threatened\" status according to IUCN Red List status), twice as much as in shrub swamp. More acutely threatened, rare species might be detected with higher sampling effort by processing more recordings. Secondary forest sites were exactly intermediary with 12 species of conservation concern, although one bird, the Javan Myna (Acridotheres javanicus), has recently been classified as vulnerable. Javan Mynas are commonly sold on the market in Jambi city. The absence of hornbills in primary forest seems fortuitous, as we also detected one Buceros rhinoceros in forest also, but at an estimated distance of 60 m. Compared to the secondary forest sites surveyed by Prabowo et al.42 in the same province (Harapan rainforest) and year, the detected bird abundance was much higher in Berbak (4 birds per count in Prabowo et al. versus 13 birds per count in the present study for the same detection radius). Our secondary forest sites seemed relatively well-preserved, as we could not detect any typical loss of understory and terrestrial insectivores due to changes in understory vegetation43. Considering that the detected abundance of birds in the other habitats was similarly high, this indicates that the National Park of Berbak provides relatively good living conditions for birds, and especially conservation-worthy species thrive in the primary forest tracts.\n\nNevertheless, bird communities differ between habitats as was shown in the non-metric multidimensional analysis (Figure S4). It turned out that the differences in bird communities arose mainly from the absence of Timaliidae in shrub swamp. Timaliidae are mainly understory insectivores, which are generally recognised to be sensitive to forest disturbance and thus act as indicator species44. The higher prevalence of generalist bulbuls and open-area kingfishers in shrub swamp is also typical of disturbed habitats in the region, such as oil palm plantations42. Notably, sunbirds were almost absent in shrub swamp, which does not seem to provide enough floral resources (pers. obs. KD). Interestingly, the species in shrub swamp were heavier, had longer wings and wider distribution ranges, indicating they may be more mobile, widespread species that are less of a conservation concern; these features are also typical of generalist species. The higher body masses may arise from the prevalence of kingfishers and bulbuls, which are relatively big.\n\n\nConclusion and outlook\n\nThe different habitats lead to a high diversity at the national park level: while the total species count per habitat was almost identical (around 50), the overall species richness reached 89 species. It is tempting to conclude that the heterogeneity introduced by disturbances such as logging or fires (which created these different habitats) increased bird species richness. However, in comparison to an equally large area consisting only of primary forest, the combination of different habitats does not lead to a markedly higher species richness, as we found a nearly identical rarefied species richness of 51 for all habitats combined. The fact also remains that primary forest harboured a higher proportion of conservation-worthy bird species, while generalists were more prevalent in the shrub swamp.\n\nOur autonomous acoustic sampling protocol gathered many more data than we analysed so far: we only processed around 0.7% of the available data, albeit the most promising dawn choruses. We would determine a higher proportion of the bird community with random or carefully chosen time windows throughout the day45. We welcome interested prospective co-authors to process more recordings to complete the species list of the still only superficially studied avifauna of Berbak. Repeated surveys to the same sites, after the major fires from 2015, would also yield insights into how bird populations are changing in the longer term in response to these and other disturbances from wildlife trade.\n\n\nData availability\n\nDataset 1: The data and R script required to reproduce our graphs and results are provided. This file contains: Berbak birds.csv contains the bird detection data; Berbak vegetation.csv contains the individual DBH values for all plots; Berbak birds analysis.R is the R script that performs the analysis and graphing. DOI, 10.5256/f1000research.13996.d19550546.\n\nThe source audio material is available at http://soundefforts.uni-goettingen.de/biosounds/collection/show/3.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was funded by the German Science Foundation (DFG) with the grant number SFB990/1.\n\n\nAcknowledgements\n\nThis study was financed by the Deutsche Forschungsgemeinschaft (DFG) in the framework of the collaborative German - Indonesian research project EFForTS (CRC990). We thank the Foreign Research Permit Ministry of Research Technology and Higher Education of Indonesia for granting the permit to KD (211/SIP/FRP/SM/VI/2012). The National Park of Berbak authorities granted us access to the Berbak swamp forest and the Zoological Society of London allowed this scientific collaboration. We acknowledge support by the German Research Foundation and the Open Access Publication Funds of the Göttingen University.\n\n\nSupplementary material\n\nTable S1: Detected bird numbers in each habitat.\n\nClick here to access the data.\n\nFigure S1: Examples of sampling sites.\n\n1: BB3 shrub swamp with burned tree stump; 2: BP2 primary swamp forest; 3: BS2: secondary swamp forest with installed sound recorder and coloured ropes for the vegetation survey.\n\nClick here to access the data.\n\nFigure S2: Distribution of tree sizes in the different habitats.\n\nWe measured the diameter at breast height (DBH) of all trees with a DBH above approximately 6.4 cm (circumference of 20 cm).\n\nClick here to access the data.\n\nFigure S3: Species richness per habitat and IUCN Red List threat status.\n\nClick here to access the data.\n\nFigure S4: Non-metric multidimensional scaling of bird communities in the different habitats.\n\nBrown represents shrub swamp, light green secondary forest, dark green primary forest.\n\nClick here to access the data.\n\n\nReferences\n\nGibson L, Lee TM, Koh LP, et al.: Primary forests are irreplaceable for sustaining tropical biodiversity. 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Publisher Full Text\n\nGiesen W, Euroconsult A: Causes Of Peat Swamp Forest Degradation In Berbak Np, Indonesia, And Recommendations For Restoration. 2004; [cited 2017 Sep 4]. Reference Source\n\nPrabowo WE, Darras K, Clough Y, et al.: Bird Responses to Lowland Rainforest Conversion in Sumatran Smallholder Landscapes, Indonesia. PLoS One. 2016; 11(5): e0154876. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLambert FR, Collar NJ: The future for Sundaic lowland forest birds: long-term effects of commercial logging and fragmentation. Forktail. 2002; 127–146. Reference Source\n\nPowell LL, Cordeiro NJ, Stratford JA: Ecology and conservation of avian insectivores of the rainforest understory: A pantropical perspective. Biol Conserv. 2015; 188: 1–10. Publisher Full Text\n\nLa VT, Nudds TD: Estimation of avian species richness: biases in morning surveys and efficient sampling from acoustic recordings. Ecosphere. 2016; 7(4): e01294. Publisher Full Text\n\nDarras K, Rahman D, Sugito W, et al.: Dataset 1 in: Birds of primary and secondary forest and shrub habitats in the peat swamp of Berbak National Park, Sumatra. F1000Research. 2018. Data Source" }	[ { "id": "31795", "date": "12 Mar 2018", "name": "Badrul Azhar", "expertise": [ "Reviewer Expertise Conservation biology" ], "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\nDarras et al. present their findings about avian biodiversity in primary swamp forest, secondary swamp forest, and shrub swamp. However, I have some major concerns regarding the way the study was conducted and little information is provide to explain the study areas.\n\n“We surveyed birds in Berbak National Park, a peat swamp situated in the province of Jambi, on the east coast of the island of Sumatra in Indonesia (Figure 1). Berbak National Park is a Ramsar site25 and an Important Bird Area26. A severe drought in 1997 facilitated forest fires which were aided by human disturbance due to natural rubber collection (Dyera costulata). The burned areas subsequently developed into shrub swamp. Tree stumps also reveal that illegal selective logging affected several areas of the park, resulting in secondary forest habitats.” Comments: Provide additional information, size area of each study area? When the secondary forest was logged? Explain about the shrub swamp here.\n\n“We recorded audible sound in all 12 sites. We used autonomous sound recorders (SM2+ recorders with SMX-II microphones, Wildlife Acoustics Inc.) and did not carry out visual surveys, since acoustic recording constitutes a valid survey method for assessing bird richness27, especially for cryptic birds in tropical forests. From February to November 2013, we sampled all three habitats at one site each month for 48 hours, starting at midnight.” Comments: I’m quite confused about the field survey because not much information is provided in the current text. Do you have enough number of spatial replicates to perform GLMMs. And with just data from 12 sites, you can confidently say the results really have answered the research questions. In addition, the sample size is small i.e. 12 sites. Why you did not move the autonomous recorders to other points or sites? You established four sites in each habitat. I think you need to use point method. For example, 30-40 points at each site. I suggest the authors to write a caveat regarding the small sample size in the discussion. The 48 hours were continuous? The sound recording method should be complemented with other sampling methods next time.\n\n“we counted and measured the diameter at breast height (DBH) of all trees with a circumference above 20 cm (diameter at breast height of ~6.4 cm) inside an area of 14 × 14 m delimited by spanning 10 m coloured ropes from the central tree where the sound recorder was attached to all cardinal directions.” Comments: Provide the summary statistics for each variable.\n\n“For all models, we used tree number, tree basal area, and habitat type as predictors.” Comments: Did you include the different sampling month into the modelling work?\n\n“We detected 379 birds overall, belonging to 90 species.” Comments: After doing bird survey for ten months, your results are not very impressive compared to conventional sampling methods. Perhaps you need to have more sites or at least 30-40 points/lines per site. How many endangered/vulnerable species were recorded?\n\nReferences Comments: The reference list is not comprehensive. Authors should search and cite recent published bird studies in tropical peat swamp forest of SEA. Some articles cited in the text are not about studies from tropical peat swamp forests.\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? 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3639", "date": "14 May 2018", "name": "Kevin Darras", "role": "Author Response", "response": "Thank you very much for your critical appraisal of our manuscript. We addressed most of the issues you raised. We added the total size of the national park to the legend of figure 1 (1476 km2). Each one of our plots was a point, and we used the birds within a 50 m radius of that point for our analysis, as explained in the methods. We stated until when the secondary forest sites were logged and the shrub bush burned: “The secondary forest plots were last subject to selective logging until 2008 for BS1, 1999 for BS2, 2003 for BS3, and 2010 for BS4. The shrub swamp sites burned in 1997, and BB2 burned yearly since.”. We provided summary statistics for the total DBH in each habitat in the Fig S2 caption: 89 m2 per ha in primary forest, 64 m2 per ha in secondary forest, and 29 m2 per ha in shrub swamp. We also clarified the data collection schedule, as this also explains why we could not systematically test for the seasonal effect in our models: “From February to November 2013, we sampled all three plots of one site each month for 48 hours, starting at midnight. It was not possible to do the survey each month, or to repeat the same site sampling sequence for the second set of recordings because of access and transportation restrictions.” Also, we did not use the sampling month as a predictor in our models as we only sampled 4 sites each month and that effect would be confounded with the spatial effect from the 4 plots being close to each other. We did not move the recorders to other sites because it would have been difficult to do so. The park is very difficult to reach and go through, and we could not do more than what we achieved in 8 months of sampling. Also, we lacked skilled ornithologists to carry out point counts and it would have been prohibitively expensive. To get an impression of the field work, you can have a look at our blog post here: https://blog.f1000.com/2018/04/10/can-hear-birds/ We now cite the study of Azhar et al. 2011 in the introduction and also in the discussion about the secondary forest value: “It is noteworthy that even extensively logged peat swamp forest can harbor large species numbers.(Azhar, B., Lindenmayer, D.B., Wood, J., Fischer, J., Manning, A., McElhinny, C., Zakaria, M., 2011. The conservation value of oil palm plantation estates, smallholdings and logged peat swamp forest for birds. Forest Ecology and Management 262, 2306–2315.).” We would like to point out that the statistical analysis has been explained: “At the count level, the species richness and abundance between habitats was modelled using generalised linear mixed effects models of the poisson family (lme4 package 37 ), with site as random variable [...]”. Since we have two counts (=data points) per site, mixed models were adequate. Also, the number of replicates was sufficient for proving our points about the functional changes observed in the bird communities of the different habitats, since our statistical analysis yielded significant P-values, even when using these stringent mixed effects models. The 48 hours were continuous indeed, and we will process more data as soon as we find people interested in doing this. 387 birds found in 8 months is not impressive per se indeed, but please bear in mind that this is the result of analysing only 0.7% of our data. We also updated these numbers by better counting of simultaneous detections, yielding a data set of 426 birds (of which 394 were used in the analysis after discarding far and unidentified birds). We added the following sentence to our discussion: “[…] due to logistical restrictions, we only surveyed four sites that were close to waterways, so that that large parts of the park remain little known. “" } ] }, { "id": "32057", "date": "04 Apr 2018", "name": "Erin M. Bayne", "expertise": [ "Reviewer Expertise Conservation biology", "bioacoustics" ], "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 \"Birds of primary and secondary forest and shrub habitats in the peat swamp of Berbak National Park, Sumatra\" by Darras et al. summarizes a survey of birds via autonomous recording units in this poorly understood ecosystem. Any information on systems such as these is valuable and providing the type of data given is useful for providing baseline information that give us an idea of what species are present and thus at risk. Thus, the data as shared is very valuable for the scientific and conservation community.\nThe introduction is fairly generic in my opinion and focuses on issues that are not the central tenet of the results that are shown. It seems that the theoretical concepts of resilience/ resistance are the actual working concepts behind this paper that would make the results better contextualized. The three habitats sampled make a nice gradient for assessing how much things are the same/ different in this system. Rather than focus on issues that you did not measure (i.e. hunting, flooding, etc) keep the introduction more centralized on the things you did measure and it would be a clearer paper.\nIn the methods there are number of very large assumptions made of the reader. I previously have read your work on measuring distance to birds from ARUs. While I find the logic of doing this reasonable, I do not remember you having this level of accuracy in distance estimation (i.e. to the metre). It seems like given the novelty of your approach that you need to provide an appendix highlighting how you do this and where this level of accuracy comes from. At a minimum I think you must let the reader know that this is an estimate and recognize there is error because I find it very unlikely that you can do this to a metre for every species. Thus, some of your birds within the sampling radius were probably past 50 metres and some birds outside 50 were likely excluded. It is likely that such error is random between habitats but you have to make this an explicit assumption.\nThe use of 20 minute point counts from the same day is very reasonable for richness and calling rates etc. But I have significant concerns about double counting of the same individual over that duration. Birds move and how a listener can track the number of individuals that occur over that interval is likely very problematic. You need to justify that this did not occur for people to believe this numerical estimates. How you analyzed abundance is unclear regardless (see below).\nIn the methods you never use the terms alpha, beta or gamma when describing richness. Yet the figures do. You need to clarify the equations used to calculate beta in particular. How beta is computed needs to be described and the results described given the figure that includes it. No measurement of beta diversity that I have read would have the range you shown in figure 2 so clarification of how is essential.\nI am somewhat confused as to the amount of replication you have. I understand there to be 12 sites. At each site you have 48 hours of data. From that you picked 20 minutes at a set time. You then repeatedly sampled one morning from each month, I think. As such you have repeated measures throughout your design. As it reads, I think your analyses using models with random effects are reasonable. What I am not clear is whether or not the ADONIS test incorporates this repeated measure component. The other issue in analyses that is unclear is you say you use the sum of the maximum number of individuals vocalizing simultaneously. Does this mean you modelled the sum of the max for all repeated surveys? If so then is this a mixed model or are you using the maximum from each count with a random effect for site? Would you not then need to control for month of the year?\nNowhere do I see rationale for using an 83% confidence interval. It is rather atypical so should be justified.\nThe discussion fails to recognize the significant limitation of this study which is replication. You only have 4 replicates for each treatment so the ability to find differences, particularly in alpha is limited. Since most of the other variables build from alpha richness it is not particularly surprising that the null models fit the best. So making a really strong statement that there is no difference is not really a solid argument. There is not a huge effect size clearly, but with 100 replicates in each habitat then I suspect you would support far more complex models and be able to draw stronger conclusions.\n\nThe discussion also goes into talking about issues of this park that are not presented with the actual results. While these things maybe happening their role in what is described as the question of concern seem like filler rather than important context from the research that was conducted.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3638", "date": "14 May 2018", "name": "Kevin Darras", "role": "Author Response", "response": "Thank you very much for your critical appraisal of our manuscript. We addressed most of the issues you raised. We did not change the focus of the introduction: hunting or poaching is not mentioned in the introduction, and flooding is only mentioned once in the introduction when enumerating the importance of peat swamp forests without further discussion. Wild bird trapping is actually mentioned in the discussion but it is central to it because it poses a direct threat to the bird populations that we surveyed. Flooding is a defining element of swamp forests that the reader needs to bear in mind. That is why we kept both of these themes in the manuscript, even though we did not measure them. We do not introduce or develop the resilience concept in this paper as the study was rather designed for documenting the bird communities and to establish their conservation value and as such, it does not address that theme satisfactorily. We think that a much more stringent selection of many more sites would be needed to address this interesting subject. Our sentence “Few studies have focused on the potential benefit of disturbances for overall landscape biodiversity apart from theoretical and modelling approaches ” was changed to “Theoretical and modelling approaches are usually used to analyse the potential benefit of disturbances for overall landscape biodiversity” to avoid implying that we will investigate that topic. We also merged that part with the previous paragraph. We clarified in the text that although bird distances were estimated to the metre, accuracy is not as high, as this led to a misunderstanding: “The distance of bird vocalisations was estimated by ear to the meter; even though the accuracy is lower, estimation error was assumed to be random. Distance estimates were made based on the call loudness in the sound recording compared to the ambient sound level and knowledge of the source sound level of each species.“ It is better to do fine-grained estimates even when accuracy is low as estimation errors should be normally distributed and averaged out in the end. Our corresponding manuscript about estimating bird detection distances is in minor revisions. We clarified how we counted individuals for our analysis of abundance. We now write “For counting bird abundance, we first derived the maximum number of simultaneously vocalising individuals in each species, and then summed these maxima over all species, leading to a conservative estimate of the number of individuals”. Thus, double-counting individuals was prevented. We did not change the statistical models description itself, as it became clearer with the new text that describes how abundance was calculated, and the existing text is straightforward: “At the count level, the species richness and abundance between habitats was modelled using generalised linear mixed effects models of the poisson family (lme4 package 37 ), with site as random variable”. We do not use month as an additional random effect as our models would then be over-fitted with one random effect per data point. We clarified what our sampling units are and now use the terms “plot” and “site” consistently throughout, as it was sometimes unclear. We also explain better when each plot was sampled: “We recorded audible sound in all 12 plots two times. […] From February to November 2013, we sampled all three plots of one site (one from each habitat) each month for 48 hours, starting at midnight.” Concerning alpha and beta richness, we now elaborate how we calculated both richness values in addition to the already cited study about additive richness partitioning: “alpha richness was the mean number of species per plot, and beta richness was defined as the total number of species in the plot’s habitat (gamma richness) minus its alpha diversity.” We clarified in the methods that the ADONIS analyses were based on data pooled from both counts, so that we only investigate the general composition of the habitats without considering seasonal effects. The rationale behind using 83% confidence intervals is now supported with a proper citation.: Krzywinski, M., Altman, N., 2013. Points of significance: error bars. Nature methods 10, 921–922. This confidence interval value corresponds to intervals that touch each other at a 5% P-value. We agree that our spatial replication is limited with only four plots per habitat. However, most ecological studies fail to satisfactorily cover the geographical ranges of the ecosystem of interest, and having more plots was not possible in our case. Moreover, we disagree with the statement that most of our measures build upon alpha richness: abundance, richness, and call activity are only correlated and one cannot replace the other. However our community-weighted means of functional traits are totally independent of richness. Instead, this is precisely the point that we make in our study: even though alpha richness is similar or indistinguishable across sites, we find significant differences in the composition and function of the respective bird communities, underlining the fact that richness (or abundance) does not tell the whole story. We made that clearer in the first paragraph of the discussion that summarises the findings: “This change in the community also became apparent in a shift to bigger and more mobile species with greater distribution ranges in shrub swamp.” As you mention, with more replicates we would be able to make finer inferences, and it is indeed our aim to maximise the use of our larger raw data set at hand with future manuscript versions. However, even with our limited data set, the differences we have found so far are statistically significant and thus solid enough to support the message we tell. Still, we added the following sentence to our discussion: “[…] due to logistical restrictions, we only surveyed four sites that were close to waterways, so that that large parts of the park remain little known. “ We would like to keep the conclusion as is since this study is focused strongly on conservation, and highlighting the threats to this national park and its animals seems to be in line with our message. To develop the wild bird trapping theme further, we added: “Wild bird trapping threatens bird populations directly (Harris, J.B.C., Tingley, M.W., Hua, F., Yong, D.L., Adeney, J.M., Lee, T.M., Marthy, W., Prawiradilaga, D.M., Sekercioglu, C.H., Suyadi, Winarni, N., Wilcove, D.S., 2016. Measuring the impact of the pet trade on indonesian birds. Conservation Biology n/a-n/a. https://doi.org/10.1111/cobi.12729), and is especially worrisome as birds from the Berbak region are increasingly traded in the caged bird market of Jambi city (pers. obs. KD).“" } ] } ]	1	https://f1000research.com/articles/7-229
https://f1000research.com/articles/7-283/v1	06 Mar 18	{ "type": "Research Note", "title": "Blood-derived extracellular vesicle proteins as potential biomarkers for the diagnosis of early ER+ breast cancer and detection of lymph node involvement", "authors": [ "Rod Tucker", "Ana Pedro" ], "abstract": "Extracellular vesicles (EV’s) are membrane surrounded structures released by different cell types and are emerging as potential therapeutic and diagnostic targets in cancer. In the present study, plasma samples derived from 7 patients with metastatic and non-metastatic ER+ (estrogen receptor positive) breast cancer (BC) were collected and their respective (EVs) isolated and the protein content analyzed by mass spectrometry and FunRich analysis. Here we report on the presence of two putative plasma EV biomarkers (which were absent in healthy controls samples) that could be used to detect early ER+ breast cancer and for those with lymph node (LN) involvement However, given the preliminar nature of the work, further investigation in a larger patient cohort is warranted to corroborate these findings. If confirmed, these biomarkers could be incorporated into simple blood test kit for the early detection of those with ER+ breast cancer and lymph node involvement.", "keywords": [ "ER+ breast cancer", "extracellular vesicles", "plasma", "biomarkers", "diagnostic", "lymph node involvement", "metastases" ], "content": "\n\nExtracellular vesicles (EVs) are membrane surrounded structures released by different cell types that are involved in cellular communication and are emerging as potential therapeutic and diagnostic targets in cancer1 as in the case of early pancreatic cancer2. Structurally, EVs are contained within a phospholipid bilayer, whose composition is very similar to the cell membrane. EVs carry proteins, nucleic acids (DNA, mRNA, and miRNA), and lipids and are classified into three types: exosomes, ectosomes (microvesicles, MVs) and apoptotic bodies.\n\nTumor-derived EVs are also critical components for preparing the tumor microenvironment because they enable tumor cells to escape from the immunological surveillance3 and help in the setting of a pre-metastatic niche for the engraftment of detached cancer cells4. Both exosomes and MVs have been extensively studied and attributed various important physiological roles in cancer5,6. For instance, EVs have been found to play an important role in every phase of cancer development from cancer initiation, invasion and metastasis7. For these reasons, EVs are potential therapeutic and diagnostic targets in cancer and EV-derived biomarkers maybe useful for predicting future metastatic development and identify metastasis sites8.\n\nER+ (estrogen receptor positive) breast cancer (BC) represents 60–80% of all BC cases9,10. Here we describe our preliminary findings exploring the role of tumour derived EVs biomarkers that could ultimately be used as part of a test kit for the detection of early ER+ BC and lymph node involvement.\n\n\nMethods\n\nPlasma samples from 4 control patients (2 adult women and 2 men) which were confirmed as not having any form of BC, ER+ BC metastases, BC1 and BC2 explants EVs, SKBC and parental BC (Lyden lab, WCM, USA). Samples CF37, CF5, CF1, CF25, CF33, CF27 and CF110 were collected at Champalimaud Clinical Centre, Portugal, as part of a study on the role of tumor-derived microvesicles and bone marrow progenitor cells as diagnostic and prognostic biomarkers in advanced BC and inflammatory BC Patients (RECI/BIM-ONC/0201/2012, FCT, Portugal). ER+ BC patient samples were selected based on their stage of disease progression – confirmed by CT-scan and surgery. EVs derived from conditioned media of cells lines SKBr3, MCF7, MDA468, MDA231 and MCF10A were also used in this study (details about these samples can be found in Table 1).\n\nThis study was approved by an Ethics Review Board at Champalimaud Foundation, Portugal. All study patients provided their written, informed consent.\n\nEV purification and analysis were performed at the Lyden lab (WCM) accordingly to Andre et al. 201611. Briefly, plasma was pelleted at 500 × g for 10 min, then the supernatant was centrifuged at 20,000 × g for 20 min. Exosomes were then harvested by centrifugation at 100,000 × g for 70 min. The exosome pellet is resuspended in PBS and collected by ultracentrifugation at 100,000 × g for 70 min. The exosome pellet is resuspended in PBS and then stored at −80°C\n\nProteomic analysis was performed at the Rockefeller University, Proteomics Center as described in Hamidi et al., 201712. Proteomic analysis was performed with the help of FunRich Program version 3. Only proteins with Mascot scores of approximately 90 or >90 were considered13.\n\n\nResults and discussion\n\nClinical data on the EVs isolated from BC patient’s plasma samples and cell lines can be found in Table 1. The method used for EV isolation also precipitates lipoproteins and immunocomplexes (IC) which are known possible contaminants14. However, samples submitted for mass spectrometry analysis showed none of the recognised contaminants and it was therefore concluded that the main EV type present in these samples is the MV.\n\nIn the two patients with early BC (Table 2a), we detected HCG1745306 isoform CRA-a, a protein from the family of alpha type haemoglobins and for the patient with lymph node involvement, we detected histone H1.2 (Table 2 a–b). HCG1745306 isoform CRA-a was only present in the two patients with early BC with Mascot scores of 3208.8 and 3966.5, respectively and absent in all controls and other patient samples.\n\nAlso, represented the Mascot scores for each protein in each sample.\n\nAlso, represented the Mascot scores for each protein in each sample.\n\nHistone H1.2 was also detected in samples from the two patients with bone metastases, a parental primary BC sample and metastatic SKBr-3, MDA468, MDA231 cell lines. However, histone H1.2 was absent from the plasma sample of a patient with multiple metastases, from the non-metastatic MCF7 cell line (a non significant mascot score) and from MCF10A cells EVs (Table 2b). These observation suggests that histone H1.2 might represent a potential marker for LN involvement and metastatic potential. Recent studies suggest histone H1.2 phosphorylation may be useful as a clinical biomarker of breast and other cancers because of its ability to recognize proliferative cell populations. Both MCF7 (expressing an allelic variant A142T) and MDA231, have a greater number of histone H1.2 phosphorylations when compared to MCF10A cell line15. Curiously, phosphorylation of histone H1.2 at S173 increases during the M phase relative to the S phase, suggesting that this event is cell cycle-dependent and may serve as a marker for proliferation of cancer cells during BC invasion16,17. Also, histone H1.2 is a novel component of the nucleolar organizer regions during mitosis18 and H1.2 depletion was observed in a human BC cell line caused cell cycle G1-phase arrest19. Indeed, a higher mitotic index (≥ 7) in primary tumors is significantly associated with LN involvement20 and higher mitotic indices accurately predict axillary LN involvement at operation.21.\n\nThese initial observations though limited in sample size, suggest the possibility that HCG1745306 isoform CRA-a could be used as a potential biomarker for the detection of early ER+ BC. Moreover, histone H1.2 might represent a marker for LN involvement. A strength of our study is that samples were drawn from those with confirmed non-metastatic and metastatic disease at different sites and so are likely to be representative patients. Nevertheless, further work in a larger cohort of patients is necessary to confirm these initial findings.\n\n\nConclusion\n\nOur early results suggest that EV derived biomarkers could represent a means of identifying both patients with early ER+ BC and those in which the disease has spread to the sentinel nodes. Confirmation of these findings in a larger patient cohort might lead to the development of an antibody-antigen based test kit which could be used to provide an instant diagnosis of BC for patients visiting their primary care physician with a suspicious lump, thereby avoid the need for mammography and invasive biopsy.\n\n\nData availability\n\nDataset 1: The mass spectrometry analysis results from all patient samples 10.5256/f1000research.14129.d19659724", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work is supported by the Foundation of Science and Technology of Portugal [RECI/BIM-ONC/0201/2012], Lyden lab (Weill Cornell Medical College, USA), Champalimaud Foundation Portugal, and Romã Laboratories Ltd.\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\nInamdar S, Nitiyanandan R, Rege K: Emerging applications of exosomes in cancer therapeutics and diagnostics. Bioeng Transl Med. 2017; 2(1): 70–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMelo SA, Luecke LB, Kahlert C, et al.: Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature. 2015; 523(7559): 177–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDos Anjos Pultz B, Andrés Cordero da Luz F, Socorro Faria S, et al.: The multifaceted role of extracellular vesicles in metastasis: Priming the soil for seeding. Int J Cancer. 2017; 140(11): 2397–2407. PubMed Abstract \| Publisher Full Text\n\nKaplan RN, Riba RD, Zacharoulis S, et al.: VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature. 2005; 438(7069): 820–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrtiz A: Not all extracellular vesicles were created equal: clinical implications. Ann Transl Med. 2017; 5(5): 111. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinciacchi VR, Freeman MR, Di Vizio D: Extracellular vesicles in cancer: exosomes, microvesicles and the emerging role of large oncosomes. Semin Cell Dev Biol. 2015; 40: 41–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKosaka N, Yoshioka Y, Fujita Y, et al.: Versatile roles of extracellular vesicles in cancer. J Clin Invest. 2016; 126(4): 1163–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBecker A, Thakur BK, Weiss JM, et al.: Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis. Cancer Cell. 2016; 30(6): 836–848. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCalhoun BC, Collins LC: Predictive markers in breast cancer: An update on ER and HER2 testing and reporting. Semin Diagn Pathol. 2015; 32(5): 362–9. PubMed Abstract \| Publisher Full Text\n\nJohnston SR, Dowsett M: Aromatase inhibitors for breast cancer: lessons from the laboratory. Nat Rev Cancer. 2003; 3(11): 821–31. PubMed Abstract \| Publisher Full Text\n\nAndré Mdo R, Pedro A, Lyden D: Cancer Exosomes as Mediators of Drug Resistance. Methods Mol Biol. 2016; 1395: 229–39. PubMed Abstract \| Publisher Full Text\n\nHamidi Z, Tejero E, Schmidt R, et al.: Identification of potential blood-derived extracellular vesicles biomarkers to diagnose and predict distant metastases in ER+ breast cancer patients. biorxiv. 2017. Publisher Full Text\n\nhttp://www.matrixscience.com/help/scoring_help.html.\n\nWitwer KW, Buzás EI, Bemis LT, et al.: Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2013; 2(1): 320360. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarshman SW, Hoover ME, Huang C, et al.: Histone H1 phosphorylation in breast cancer. J Proteome Res. 2014; 13(5): 2453–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen Y, Hoover ME, Dang X, et al.: Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence. Mol Cell Proteomics. 2016; 15(3): 818–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen, et al.: Characterization of Histone H1 Phosphorylated Proteoforms during Breast Cancer Invasion, to appear in Mol Cell Proteomics.. 2015.\n\nChen J, Teo BHD, Cai Y, et al.: The linker histone H1.2 is a novel component of the nucleolar organizer regions. J Biol Chem. 2018; 293(7): 2358–2369. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSancho M, Diani E, Beato M, et al.: Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth. PLoS Genet. 2008; 4(10): e1000227. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIllyes I, Tokes AM, Kovacs A, et al.: In breast cancer patients sentinel lymph node metastasis characteristics predict further axillary involvement. Virchows Arch. 2014; 465(1): 15–24. PubMed Abstract \| Publisher Full Text\n\nAaltomaa S, Lipponen P, Eskelinen M, et al.: Nuclear morphometry and mitotic indexes as prognostic factors in breast cancer. Eur J Surg. 1991; 157(5): 319–24. PubMed Abstract\n\nRen W, Liu Y, Wan S, et al.: BMP9 inhibits proliferation and metastasis of HER2-positive SK-BR-3 breast cancer cells through ERK1/2 and PI3K/AKT pathways. PLoS One. 2014; 9(5): e96816. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBonnomet A, Syne L, Brysse A, et al.: A dynamic in vivo model of epithelial-to-mesenchymal transitions in circulating tumor cells and metastases of breast cancer. Oncogene. 2012; 31(33): 3741–53. PubMed Abstract \| Publisher Full Text\n\nTucker R, Pedro A: Dataset 1 in: Blood-derived extracellular vesicle proteins as potential biomarkers for the diagnosis of early ER+ breast cancer and detection of lymph node involvement. F1000Research. 2018. Data Source" }	[ { "id": "32046", "date": "26 Mar 2018", "name": "Shweta Aras", "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 research note by Tucker et al., authors have identified 2 novel biomarkers potentially involved in early breast cancer development as well as as lymph node metastasis using extracellular vesicles from patient samples along with adequate controls of metastatic and non-metastatic cell lines. Several reports in the literature have already suggested the importance of EVs in cancer initiation and progression in various types of cancers. Although the novelty of this publication is limited, identification of 2 new molecules differentially expressed in primary versus metastatic breast cancer patients opens up the possibility of them being used in a simple prognostic blood test for detecting early BC development as opposed to mammography and other invasive techniques. A probable limitation of the study would be lack of identification of molecular mechanisms through which this upregulation of CRA-a and histone H1.2 phosphorylation plays a role in metastasis, specifically in which step of metastatic cascade e.g. intravasation, EMT, extravasation etc? But overall, the authors have done a great job and the article is a good addition to the 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? 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? 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? Yes", "responses": [] }, { "id": "32581", "date": "16 Apr 2018", "name": "Matthew J. Shurtleff", "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\nTucker and Pedro present results from proteomics studies of material precipitated from the plasma of breast cancer patients and healthy controls, and from the media of explant cultures and cell lines. They observe CRA-a (HBA2, alpha hemoglobin) as being present in precipitated material from the plasma of patients with early breast cancer and Histone H1.2 as being present in samples with lymph node involvement (plasma), and the media of explant cultures of bone metastases and metastatic cell lines.\nWhile the results are clearly presented, the interpretation that these are EV-based biomarkers is not supported. The EV isolation method used is insufficient to purify EVs from other contaminants. Furthermore, it is not intuitive that EVs would be likely to contain a histone (normally confined to the nucleus) or hemoglobin. The authors' discussion of this major concern is insufficient (\"However, samples submitted for mass spectrometry analysis showed none of the recognised contaminants and it was therefore concluded that the main EV type present in these samples is the MV.\").\nThe EV literature is quite confusing due, in part, to the over-interpretation of observations from samples prepared using inadequate isolation methods. Therefore, in it's current version, I do not recommend indexing of this report. Ideally, the authors should repeat the work with approaches that better enrich for EVs over non-EV protein contaminants (e.g. density-gradient based ultracentrifugation or immuno-affinity purification) and protease protection assays in the presence/absence of detergent should be used evaluate if the proteins are indeed associated with EVs and if they are on the surface or interior of vesicles. If further experiments are not possible, I recommend revising the paper to remove the unsupported conclusion that the proteins identified are EV-associated. (For example the title could be: Blood-derived extracellular proteins as potential biomarkers for the diagnosis of early ER+ breast cancer and detection of lymph node involvement).\nFor reference, I direct the authors to an updated publication prepared by members of the EV community on the minimal information for studies of EVs in the Journal of Extracellular Vesicles1.\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? 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": [] } ]	1	https://f1000research.com/articles/7-283
https://f1000research.com/articles/7-562/v1	09 May 18	{ "type": "Case Report", "title": "Case Report: A Primordial odontogenic tumor", "authors": [ "Hatem Wael Amer", "Layla Hafed", "Sally Ibrahim", "Hatem Wael Amer", "Sally Ibrahim" ], "abstract": "Introduction: Primordial odontogenic tumors are a rare recently described mixed odontogenic tumor composed histopathologically of dental papilla like tissue and enamel organ like tissue. Only nine cases have been documented worldwide and we are reporting the tenth case which is from Egypt. Clinical finding: A 2-year-old Egyptian boy that presented with an asymptomatic swelling of the mandible which appeared with multilocular radiolucency associated with an impacted developing tooth on a computerized tomography (CT) scan. Diagnoses, interventions, and outcomes: The lesion was excised and diagnosed as a primordial odontogenic tumor. The patient was followed up for two years with no recurrence. Conclusion: Differentiation of primordial odontogenic tumors from other odontogenic tumors, which resemble it histopathologically is crucial to avoid unnecessary aggressive treatment.", "keywords": [ "Primordial", "Mixed odontogenic tumor", "Jaw tumor", "Odontogenic." ], "content": "Introduction\n\nPrimordial odontogenic tumor (POT) is a recently described mixed odontogenic tumor described in the last WHO classification of head and neck tumors1. This tumor has been described as other entities in the past, because of its histological similarity to other odontogenic tumors as ameloblastic fibroma, odontogenic myxoma, and odontogenic fibroma and hyperplastic dental follicles2.\n\nMosqueda-Taylor et al.3 described and denominated this novel lesion which did not fulfil the criteria of any of the previously classified odontogenic tumors by reporting the clinicopathological and immunohistochemical features of six cases diagnosed as primordial odontogenic tumor.\n\nPrimordial odontogenic tumors are characterized histologically by a variably cellular loose fibrous tissue with areas similar to the dental papilla, covered by cuboidal to columnar epithelium that resembles the internal epithelium of the enamel organ, surrounded at least partly by a delicate fibrous capsule1.\n\nOnly nine cases have currently been reported, and we report an additional case of an primordial odontogenic tumor from Egypt.\n\n\nCase report\n\nA 2-years-old Egyptian boy referred to Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Cairo University in November 2015 with a fleshy swelling arising from site of marsupialization performed two months previous. By taking patient’s history we established the lesion arose as a painful swelling covered with normal mucosa causing obliteration of the vestibule with two months duration. Manual examinations of the regional lymph nodes were negative on examination. By computerized tomography (CT) scan, a multilocular radiolucent lesion was seen associated with an impacted developing tooth in the mandibular posterior area measuring 3cm × 4cm (Figure 1).\n\nOn aspiration, straw cystic fluid was noted. Complete surgical excision of the lesion with the impacted tooth was performed. And the excised lesion was sent to the Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Cairo University. The gross specimen showed a cystic lesion which showed areas of thickening. Hematoxylin and eosin stained sections revealed surface columnar and cuboidal epithelium covering a loose and myxoid fibrous tissue (Figure 2) and this specimen was diagnosed as a primordial odontogenic tumor. The patient was followed up for two years with no recurrence, and new bone formation was detected in the follow up radiographs (Figure 3).\n\n\nDiscussion\n\nPOT is a new entity first reported in a case series of 6 cases in 2014 described as benign mixed odontogenic tumor by Mosqueda-Taylor et al.3. Then another two cases were reported in 2015 and 2017 by Slater et al.4 and Ando et al.5 respectively then in 2018 Bajpai and Pardhe6 described another case. This novel lesion was added to the new WHO classification of odontogenic tumors1.\n\nTable 1 shows the clinicopathological and radiographic data of the nine documented cases. All reported patients were of young age group, ranging from 3–19 years with almost equal sex predilection and with posterior mandible as predominate site. All of these clinical finding are similar to this reported case.\n\nM: Male; F: Female; RL: Radiolucent; UL: Unilocular; ML: Multilocular; mm: millimeter.\n\nAll reported lesions were expansile and asymptomatic which are opposite to our case as it was painful during presentation, which may be the result of the previous marsupialization.\n\nRadiographically, POT presents with a well-defined radiolucent lesion, either unilocular or bilocular, except in the case of Bajpai and Pardhe6 who reported a case that appeared mulitlocular.\n\nAll documented cases shared similar histopathological criteria proposed by Mosqueda-Taylor et al.3, as did our present case, where loose and myxoid connective tissue stroma resembles the dental papilla covered by columnar epithelium of a single layer, with the epithelium resembling the inner enamel epithelium.\n\nRegarding the treatment approach, all previously documented lesions were treated with enucleation with different periods of follow up and reported no recurrence, in line with our presented case.\n\nIn conclusion, this is the first report case of POT from Egypt after it was defined in the latest WHO classification. Differentiation between POT and other closely resembling odontogenic tumors is crucial, especially in the case of odontogenic myxomas as it is a more aggressive tumor and requires more aggressive treatment.\n\nThe clinical, radiographical and histopathologic data of the nine previously documented cases in addition to our case will be useful to differentiate this new tumor from other odontogenic tumors, which resemble it histopathologically, to avoid unnecessary aggressive treatment modalities.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and images was obtained from the patient's parent.\n\n\nData availability\n\nDataset 1: Raw histological image. A photomicrograph of Hematoxylin and eosin (H&E) stained sections showing primitive connective tissue stroma covered by columnar epithelium, (×200). 10.5256/f1000research.14735.d2022147", "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\nEl-Naggar AK, Chan JK, Grandis JR, et al.: WHO Classification of Head and Neck Tumours. 4th edition. Lyon: IARC Press, 2017. Reference Source\n\nIde F, Kikuchi K, Kusama K, et al.: Primordial odontogenic tumour: is it truly novel? Histopathology. 2015; 66(4): 603–4. PubMed Abstract \| Publisher Full Text\n\nMosqueda-Taylor A, Pires FR, Aguirre-Urizar JM, et al.: Primordial odontogenic tumour: clinicopathologic analysis of six cases of a previously undescribed entity. Histopathology. 2014; 65(5): 606–12. PubMed Abstract \| Publisher Full Text\n\nSlater LJ, Eftimie LF, Herford AS: Primordial Odontogenic Tumor: Report of a Case. J Oral Maxillofac Surg. 2016; 74(3): 547–51. PubMed Abstract \| Publisher Full Text\n\nAndo T, Shrestha M, Nakamoto T, et al.: A case of primordial odontogenic tumor: A new entity in the latest WHO classification (2017). Pathol Int. 2017; 67(7): 365–9. PubMed Abstract \| Publisher Full Text\n\nPardhe N, Bajpai M: Primordial Odontogenic Tumor of Mandible; A Case with Proposed Diagnostic Criteria. Iran J Med Sci. 2018; 43(1): 97–99. PubMed Abstract \| Free Full Text\n\nAmer H, Hafed L, Ibrahim S: Dataset 1 in: Case Report: A Primordial Odontogenic Tumor. F1000Research. 2018. Data Source" }	[ { "id": "34186", "date": "23 May 2018", "name": "Eman Abdelzaher", "expertise": [ "Reviewer Expertise Neuropathology", "Surgical Pathology" ], "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-written case report describing a rare entity which was recently described. I would encourage the authors to add more histopathological details and elaborate on the discussion section mainly focusing on important differentiating points with histopathological mimics.\nPlease click here for the annotated PDF of this article.\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": "3896", "date": "13 Aug 2018", "name": "Layla Hafed", "role": "Author Response", "response": "Thank you doctor for your approval. We mentioned all the histopathological criteria that were reported in the last WHO classification of head and neck tumors which are pathognomonic for POT where the columnar epithelium covering the primitive connective tissue stroma could not be seen in any other histopathological mimics." } ] }, { "id": "34185", "date": "05 Jun 2018", "name": "Marwa Mokbel ElShafei", "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 presented here is a well constructed case, the rarity and novelty of the lesion makes it useful for the clinicians and indeed, as stated, it may lead to a more conservative treatment and save the patient an aggressive treatment. The young age of the patient is interesting and gives a probable reassurance for the parents as only a conservative treatment is needed. A clinical picture for the patient would have been useful if present and also a clinical picture after full recovery. A need to switch the magnification on the histopathologic pictures is to be done.\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": "3895", "date": "13 Aug 2018", "name": "Layla Hafed", "role": "Author Response", "response": "Thank you doctor for your approval. Unfortunately, the surgeon did not take photos for the patient. And about the numbers on the histopathologic pictures those are the measure of the microscope scale bar not the magnification." } ] }, { "id": "34183", "date": "05 Jun 2018", "name": "‎Eman A. Abo Hager", "expertise": [ "Reviewer Expertise Oral and dental pathology" ], "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 case report describing in a full details a rare recently described mixed odontogenic tumor. I would prefer the authors to suggest further diagnostic test to differentiate peimordial odontogenic tumor from the aggressive odontogenic myxomas especially when cases unexpectedly appear radiographically multilocular radiolucent as in the present case.\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": "3891", "date": "09 Aug 2018", "name": "Layla Hafed", "role": "Author Response", "response": "A panel of epithelial and mesenchymal IHC markers could be done but they are of no valuable diagnostic benefits as recorded by previous studies. Careful H & E sections examination is enough, the present of columnar epithelium covering the primitive connective tissue stroma could differentiate the POT from the odontogenic myxoma which lacks the covering." } ] }, { "id": "34184", "date": "12 Jul 2018", "name": "Manal Al-Hajri", "expertise": [ "Reviewer Expertise oral medicine and periodontologiest" ], "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 article provides a good idea about POT especially because they are rare tumors and only a few cases have been reported. Article also writes all diagnosis methods and treatment outcomes and compared all cases of POT. Follow up of cases, I think, need a longer period than two years to decide no recurrence.\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": "3890", "date": "09 Aug 2018", "name": "Layla Hafed", "role": "Author Response", "response": "Sure doctor, two years of followup are not enough to decide no recurrence. For that the patient is still under followup for the third year and there is no recurrence." }, { "c_id": "3894", "date": "09 Aug 2018", "name": "Layla Hafed", "role": "Author Response", "response": "Thank you doctor for your approval." } ] } ]	1	https://f1000research.com/articles/7-562
https://f1000research.com/articles/7-559/v1	08 May 18	{ "type": "Research Article", "title": "In vitro evaluation of the antifungal activity of Marco (Ambrosia arborescens Mill.) and Matico (Aristeguietia glutinosa Lam.) on the pathogenic fungi that cause dermatomycosis (ringworm)", "authors": [ "Tatiana de los Ángeles Mosquera Tayupanta", "Sandra Elizabeth Ayala Valarezo", "Tatiana Alexandra Vasquez Villareal", "María Belén Montaluisa Álvarez", "Sandra Elizabeth Ayala Valarezo", "Tatiana Alexandra Vasquez Villareal", "María Belén Montaluisa Álvarez" ], "abstract": "Background: Currently, there is a trend towards using natural and ethnopharmacological species with therapeutic potential. This investigation evaluated the antifungal activity of two species in the Ecuadorian Andes, which are used in treating dermatomycosis: Ambrosia arborescens Mill. (Marco) and Aristeguietia glutinosa Lam. (Matico). Methods: We worked with seven concentrations (100 to 700ppm) of Ambrosia arborescens Mill. extract and ten concentrations (0.5 to 5%) of essential oil (EO) of Aristeguietia glutinosa Lam. on Trichophyton mentagrophytes ATCC 9533, Trichophyton rubrum ATCC 28188, Microsporum canis ATCC 36299 and Candida albicans ATCC 10231. The methodology used was a modified version of the Kirby-Bauer method, using diffusion in agar wells. Results: The Tukey test, after the one-way Anova, determined effective concentrations of EO: 5% for Trichophyton mentagrophytes, 4.5% for Trichophyton rubrum, 5% for Microsporum canis and 2% for Candida albicans. In the extracts, the concentration of 700ppm was used for Trichophyton mentagrophytes, Trichophyton rubrum, and 600ppm for Microsporum canis and Candida albicans. Conclusions: The evaluation of the antifungal activity of the Ambrosia arborescens extract showed inhibition in the studied dermatophytes in each one of the planted concentrations (100 to 700ppm). The evaluation of the antifungal activity of Aristeguietia glutinosa EO showed inhibition in the studied dermatophytes in each of the planted concentrations (0.5 to 5%).", "keywords": [ "Ambrosia Arborescens", "Aristeguietia glutinosa", "essential oil", "extract", "antifungal", "dermatomycosis" ], "content": "Introduction\n\nThis research provides a technical basis that allows for plants used in the field of ethnomedicine to be used in the development of natural products in concentrations that allow for an effective therapeutic action. Plants have been identified as being useful in treating several diseases that have affected populations since ancient times, amongst which are allergies, pains, infections and skin diseases. The most studied skin lesions are related to different forms of dermatitis, skin allergies, acne and bacterial infections of the skin. Several medicinal plants, or essential oils and natural extracts obtained from them, have a curative influence on these pathologies1.\n\nMany plants have been granted the classification of \"medicinal\", being part of the intangible natural heritage of a country, thus it is advisable to take advantage of ancestral knowledge2 in the development of new therapeutic agents. Current knowledge about medicinal plants has two fundamental objectives, once their therapeutic value has been validated. The first is to discover the molecular structure of the active principles and the second is to lay down scientific bases for the direct use of the plants by large sectors of the population3. Ambrosia arborescens (Marco) and Aristeguietia glutinosa (Matico) belong to the Asteraceae family, which are characterised as being aromatic plants with medicinal properties. Marco is widely used amongst the inhabitants of our region in order to mitigate headaches, migraines, rheumatism, fever, constipation, prostate disorders, fractures and injuries as well as for vaginal baths4. Within agriculture, it is used as a biological means of controlling pests, related to the protection or defense against attacks of microorganisms and insects5. Matico is a native species of the Inter-Andean valley and is found only in Ecuador. It is attributed with having anti-inflammatory, expectorant, antitussive, healing and disinfectant properties. It is also used as an emollient and protector of the skin6.\n\nIn the same way that the nutritional value of certain plant products has been discovered over the years, humankind has also determined the curative or analgesic value of many plants. The extinction of plants and loss of cultural knowledge puts at risk new applications which have not yet been developed by modern science and might be valid, safe and effective5.\n\nSynthetic drugs offer indisputable advantages, however it has been found that undesirable and even serious sideeffects can occur. This has encouraged a return to the study of plants in an attempt to find natural drugs that have higher therapeutic value and lower pathological risks. Although empirical knowledge is valuable, it is not reliable enough, thus scientific confirmation is necessary3. In the case of dermatoses, the most commonly used medication for lesions is Clotrimazole (a compound derived from the imidazole family) commonly used for the treatment of yeast infections, oral candidiasis, and dermatophytosis (ringworm). It is also used to treat athlete’s foot and tinea corporis7.\n\nAnother problem is the innate and acquired resistance that has been demonstrated in vitro and in vivo, therefore the early recognition of species resistant to one or more antifungals guarantees an optimal treatment and better result. However, resistance is constantly growing and evolving, so the introduction of new antifungals is important8.\n\nThe fungi that produce superficial mycoses are opportunistic, thus patients with one or more of the following are more susceptible: diabetes, AIDS, cancer and any other debilitating or chronic condition. Worldwide, a prevalence of 5% to 10% of mycosis caused by Candida has been found by dermatology services. Other isolated species with greater frequency are Tinea corporis and Tinea capitis as well as the following fungi: Microsporum canis (9%), Trichophyton mentagrophytes (8.8%) and Trichophyton rubrum (8.6%)9.\n\nThe ethnobotanical use of species such as Marco (Ambrosia arborescens) and Matico (Aristeguietia glutinosa) in skin conditions allows them to be considered inhibitory agents of pathogenic fungi. The verification of this activity in vitro allowed us to determine the most effective concentrations and consequently one could standardise the use of these species as natural assets in the formulation of products for the treatment of dermatomycosis.\n\n\nMethods\n\nThe vegetal material was collected in Ibarra City in the province of Imbabura. Ambrosia arborescens was obtained from the surrounding area of the Yaguarcocha Lagoon and Aristeguietia glutinosa in the San Miguel de Arcángel neighbourhood on the Alto de Reyes Hill. Ibarra is 2225 metres above sea level (m.a.s.l.). Located 115km northwest of Quito and 125km south of the city of Tulcán, it has a temperate and pleasant dry climate. The historical meteorological annuals have determined an average temperature of 15, 90°C, with a minimum variation of less than 0, 3°C.. Records show a maximum mean temperature of between 20 and 25°CC and a minimum mean of between 7 and 11°CC (see Table 1). Maximum average wind speeds are 7m/s and the minimum are 3.5 m/s. A hydrometeorological analysis has determined that average annual precipitations are between 1000 mm and 1400 mm10.\n\nData was obtained on the day of collection is presented in Table 1.\n\nThe material was dried under referenced conditions that guarantee not to affect it, namely that air temperature should be moderated to avoid the temperature of the herbs exceeding critical temperature (generally between 30 and 60°C for medicinal plants). Drying interrupts the degradation processes caused by enzymes or ferments and prevents the development of microorganisms and oxidation and hydrolysis reactions11.\n\nOf all the fungal population, with regard to the group of fungi and complying with international regulations, depending on the incidence of skin diseases9, the in vitro analysis was carried out on the following microorganisms: Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Candida albicans, which are fungi with high influences on treated dermatomycoses.\n\nWe worked with two types of extracts. We used a soft extract for Marco (Ambrosia arborescens), obtained by placing percolation in 96% ethyl alcohol (1:4) for 72 hours at room temperature, with a subsequent evaporation in a vacuum until a consistency of thick stringy mass was achieved (15 to 25% humidity) and the concentration is 2:1 according to Osorio (2009)12. In the case of Matico (Aristeguietia glutinosa), a steam distillation at 92°C was carried out in a distiller for 45 minutes until the essential oil was obtained.\n\nThe method is based on the relationship between the concentration of the substance needed to inhibit a bacterial strain and the growth inhibition halo on the surface of an agar plate with a suitable culture medium, planted homogeneously with the bacteria to be tested and upon which a 6mm diameter filter paper disc has been placed, or planted in an impregnated well with the correct amount of the substance13. The Microbiologics-KWIK-STIKTM strains Trichophyton mentagrophytes ATCC 9533, Trichophyton rubrum ATCC 28188, Microsporum canis ATCC 36299 and Candida albicans ATCC 10231 were used for the evaluation. We carried out an activation of the lyophilized strains and a subsequent culture in the first three microorganisms. We prepared a suspension of spores, evaluating the concentration of them with the Neubauer count chamber and the planting value or inoculum for the three microorganisms was 106. In the case of Candida albicans ATCC 10231, the preparation of the inoculum was carried out by means of the method for the quantification of the growth of microbial populations based on the cell mass by tubidimetry using a UV Shimadzu Spectrophotometer (model: mini 1240). The bibliographically referenced conditions determine a wavelength of 625 nm, an absorbance value of 0.08 to 0.11 which is equivalent to 1-2x108 UFC ml for bacteria and a wavelength of 530nm and absorbance value of 0.13 to 0.16 which is equivalent to 1-5x106 cells for fungi and yeasts14. Seven successive dilutions of Ambrosia arborescens (Marco) extract. Concentrations of 100ppm, 200ppm, 300ppm, 400ppm, 500ppm, 600ppm and 700ppm (parts per million) were made using dimethyl sulphoxide (DMSO) polar miscible fluid and without antifungal activity as diluent. 10 dilutions of Aristeguietia glutinosa (Matico) essential oil were made in percentages of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% and 100% using Dimethylsulfoxide (DMSO) as the diluent. Planting was executed in plates by mass homogenisation for Trichophyton mentagrophytes, Trichophyton rubrum and Microsporum canis. 1ml of inoculum of the fungus was deposited and 19 ml of molten Sabouraud Dextrose Agar (SDA) culture medium was added, tempered at approximately 45°C and sterile. Both were mixed by gentle rotation of the plates. In this way, the microorganisms were distributed homogeneously in the culture medium allowing growth throughout the agar15.\n\nOnce the inocula were planted, the natural extracts were placed in the indicated concentrations following the Kirby-Bauer methodology, a method which was modified for use with diffusion in agar wells. The wells were made on the surface of the agars with the help of a sterile hole puncher with a diameter of 6mm. Then, 50µl of different concentrations of extract, different concentrations of oil, as well as a positive blank (clotrimazole) and negative blank (Dimethylsulfoxide-DMSO) were poured into each of them. The plates were incubated for 2 to 5 days at 25°C to allow confluent fungal growth. All tests were done in triplicate. The inhibition halos were measured with a gauge or calliper (mm), considering them as starting from the point of complete inhibition as seen by the naked eye. The reading was taken on the back of the plate with a black background and reflected light. The interpretation of the reading was based on the Kirby-Bauer method, which relates the concentration of the substance necessary to inhibit a bacterial strain with the growth inhibition halo on the surface of an agar plate with a suitable culture medium and seeded homogeneously with the tested fungi and on which has been deposited a (6 mm diameter) filter paper disk16.\n\nThe statistical analysis was carried out with a two-way ANOVA and the Tukey Test (HSD) with the use of the Statistix 10.0 General AOV/AOCV Program and the Statistix 10.0 Tukey HSD All-Pairwise Comparisons Test Program.\n\n\nResults\n\nTable 2 shows the results of an average of three determinations whose deviation limit is close to 95%. The results showed antifungal activity in each of the concentrations and the positive blank (Clz) used on Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Candida albicans.\n\nTable 3 shows the results of an average of three determinations whose limit of deviation is close to 95%. The results evidenced antifungal activity in each of the concentrations and the positive blank (Clz) used on Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Candida albicans.\n\nThe two-factor ANOVA statistical analysis resulted in a probability of p=0.0004 which is less than α (0.05). The statistical test F is 8.23, higher than the Ft of 5.41. These values allow one to accept the alternative hypothesis, which is that Matico (Aristeguietia glutinosa Lam.) EO presents antifungal activity (Figure 1). Once the alternative hypothesis was accepted, the Tukey test was carried out a posteriori to demonstrate whether or not there is a significant difference between the inhibition halos.\n\nThe test evidenced the existence of two groups: A and B. Group A consisted of Microsporum canis and group B consisted of Candida albicans, Trichophyton rubrum and Trichopyton mentagrophytes. The most evident inhibition average was seen on Microsporum canis with an average of 20.81mm (Table 4).\n\nThe two-factor ANOVA statistical analysis resulted in a probability of p=0.000 which is less than α(0.05) and the statistic of the F test is 24.16, higher than the Ft of 4.76. These values allow one to accept the alternative hypothesis, which is that extract of Marco (Ambrosia arborescens Mill.), presents antifungal activity (Figure 2). Once the alternative hypothesis was accepted, the Tukey test was carried out a posteriori to show whether or not there is a significant difference between the inhibition halos.\n\nThe test evidenced the existence of three groups: A, B and C. Group A consisted of Microsporum canis and Trichophyton rubrum, group B of Trichophyton rubrum and Trichopyton mentagrophytes and group C of Candida albicans. The most evident inhibition average was seen on Microsporum canis with an average of 31.20mm (Table 5).\n\n\nConclusions\n\nThe evaluation of the antifungal activity of the Ambrosia arborescens extract showed inhibition in the studied dermatophytes in each one of the planted concentrations (100 to 700ppm). The data discrimination of the Tukey test, after the one-way Anova, showed that the most effective concentrations were at 700ppm for Trichophyton mentagrophytes and Trichophyton rubrum and 600ppm for Microsporum canis and Candida albicans. The two-way Anova statistical analysis proved that the extract presented a greater homogeneity at concentrations of 300 and 400ppm over the studied dermatophytes in comparison to the other concentrations, obtaining an average halo of 23.37mm. The evaluation of the antifungal activity of Aristeguietia glutinosa EO showed inhibition in the studied dermatophytes in each of the planted concentrations (0.5 to 5%). The data discrimination from the Tukey test, after the one-way Anova, showed that the most effective concentrations were 5% for Trichophyton mentagrophytes, 4.5% for Trichophyton rubrum, 5% for Microsporum canis and 2% for Candida albicans. The two-way ANOVA (analysis of variance) proved that the EO has a greater uniformity of inhibition at 2, 2.5 and 3% in relation to the other concentrations, for which an average halo of 15.75mm was determined on the studied dermatophytes.\n\n\nData availability\n\nDataset 1. Diameter of inhibition halos of Ambrosia arborescens extract. Diameter (mm) of inhibition halos of Ambrosia arborescens EO. positive control (Clz +) and negative control (DMSO -) used on Trichophyton mentagrophytes. Trichophytonrubrum. Microsporum canis. Candida albicans. 10.5256/f1000research.14354.d20149217\n\nDataset 2. Diameter of inhibition halos of Aristeguietia glutinosa essential oil against fungus. Diameter (mm) of inhibition halos of Aristeguietia glutinosa essential oil. positive control (Clz +) and negative control (DMSO -) used on Trichophyton mentagrophytes. Trichophyton rubrum. Microsporum canis. Candida albicans. 10.5256/f1000research.14354.d20149318", "appendix": "Author contributions\n\n\n\nMatteo Radice confirms that the author has an appropriate level of expertise to conduct this research, and confirms that the submission is of an acceptable scientific standard. Matteo Radice declares they have no competing interests. Affiliation: Faculty of Agroindustry and Faculty of Agricoltural and Zootechnics, Universidad Estatal Amazonica, Puyo, Ecuador.\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\nCentro Nacional de Investigaciones para la Agroindustrialización de Especies Vegetales Aromáticas y Medicinales Tropicales: Aplicaciones de los aceites esenciales y compuestos naturales en productos cosmecéuticos para el cuidado de la piel. Bogotá: COLCIENCIAS. 2008. Reference Source\n\nTapia W, Garzón K, Granda N, et al.: Alexiteric activity of Costus pulverulentus C. Presl., Desmodium adscendens (Sw.) DC., Begonia glabra Aubl. and Equisetum bogotense on the poison of Bothrops asper (equis) [version 1; referees: 1 not approved]. F1000Res. 2018; 7: 136. Publisher Full Text\n\nChiriboga X: Validación de plantas medicinales del Ecuador. QUITO: Abya-Yala. 2010.\n\nDe la Torre L, Navarrete H, Muriel P, et al.: Enciclopedia de las Plantas útiles del Ecuador. Quito: Aarhus. 2008. Reference Source\n\nNaranjo P: La Etnomedicina en el Ecuador. Etnomedicina y etnobotánica: avances en la investigación. 2010; 61–71.\n\nVarela J, Lavaggi M, Cabrera M, et al.: Fraccionamiento bioguiado del extracto hidro-etanólico de Aristeguietia glutinosa Lam, y elucidación estructural de los principios activos anti-Trypanosoma cruzi. Uruguay: Universidad de la República de Montevideo. 2011. Reference Source\n\nFDA: Clotrimazole official FDA information,side effects and uses. Sellersville. 2011.\n\nLass-Flörl C, Perkhofer S, Mayr A: In vitro susceptibility testing in fungi: a global perspective on a variety of methods. Mycoses. 2010; 53(1): 1–11. PubMed Abstract \| Publisher Full Text\n\nVillares MP: Identificación de hongos asociados a infecciones dérmicas en pacientes diabéticos tipo II que acuden al Hospital Provincial Docente Ambato Junio-Noviembre 2010. Tesis Universidad Técnica de Ambato, Tungurahua, Ecuador. 2012. Reference Source\n\nNaranjo M, Dávalos M, Batallas B, et al.: Proyecto analisís de vulnerabilidades a nivel municipal: perfil territorial cantón San Miguel de Ibarra. Cotopaxi: Universidad Técnica del Norte. 2013. Reference Source\n\nSharapin N: Fundamentos de Tecnología de Productos Fitoterapéuticos. Bogotá: Convenio Andrés Bello. 2000. Reference Source\n\nOsorio E: Aspectos básicos de farmacognosia. Antoquia: Universidad de Antioquia. 2009. Reference Source\n\nNational Committee for Clinical Laboratory Standars: Prueba de suceptibilidad antimicrobiana por difusión en agar. 1997. Reference Source\n\nRosato A: Microbiologia Farmaceutica Seconda Edizione. Napoli: EdiSES. 2013. Reference Source\n\nQuiroz M: Manual de procedimiento de laboratorio docente de microbiología clínica en base a la normativa ISO 9001: 2008. Quito: Universidad Central del Ecuador Facultad de Ciencias Químicas. 2012. Reference Source\n\nRamirez LS, Marin DM: Metodología para evaluar in vitro la actividad antibacteriana de compuestos de origen vegetal. Red de Revistas Científicas de América Latina, el Caribe, España y Portugal. 2009; 15(42): 263–268. Reference Source\n\nde los Ángeles Mosquera Tayupanta T, Ayala Valarezo SE, Vasquez Villareal TA, et al.: Dataset 1 in: In vitro evaluation of the antifungal activity of Marco (Ambrosia arborescens Mill.) and Matico (Aristeguietia glutinosa Lam.) on the pathogenic fungi that cause dermatomycosis (ringworm). F1000Research. 2018. Data Source\n\nde los Ángeles Mosquera Tayupanta T, Ayala Valarezo SE, Vasquez Villareal TA, et al.: Dataset 2 in: In vitro evaluation of the antifungal activity of Marco (Ambrosia arborescens Mill.) and Matico (Aristeguietia glutinosa Lam.) on the pathogenic fungi that cause dermatomycosis (ringworm). F1000Research. 2018. Data Source" }	[ { "id": "33805", "date": "21 May 2018", "name": "María-Elena Cazar", "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\nTo be appropriate, this scientific work and the manuscript produced needs important improvements. The authors do not explain the reason why an organic extract from A. arborescens and an essential oil from A. glutinosa are studied. The results are not compared with the current knowledge of the plant species selected. In scientific literature there are hundreds of references regarding the bioactivity and secondary metabolites production from A. arborescens and A. glutinosa. The authors apply a well-known method to assay the antifungal activity. I strongly recommend to review the guidelines provided by the CLSI (Clinical and Laboratory Standards Institute) to set the conditions to perform an antifungal assay. In this document, the standard conditions to evaluate antifungal agents are clearly stated, including inoculums concentration and dilutions range for antimicrobial agents.\nThe introduction of the manuscript needs revisions. This sections needs to highlight the valuable information resulted from this work. The relevance of medicinal plants for traditional medicine, the side effects of current antifungal agents are well known. The first paragraph must be at the end of this section, presented according to a frame constructed from the current knowledge of plant-based antifungal agents.\nIn the Methods sections there are several bulk mistakes\nVegetal material: “plant material” is correct, rather than “vegetable material”. The geographic (not geomorphologic) data from the collection sites of the plant material are not suitable to be presented in a table. Besides, the data are repeated in the text and the table. The consideration for drying conditions are confusing.\n\nFungal population. The use of this title is completely wrong. I guess the authors tried to state the fungal strains used for the bioassay. In this section the ATCC codes must be provided.\n\nObtaining extracts: Please change this title as well. In this work one plant extract and one essential oil were obtained from the plants studied. Start with the plant extract preparation. I assume the authors preparing the extract contacting the plant material with 96% ethanol. “placing percolation” is not correct. Do not use the first-person plural for writing this section (this advice applies for all the manuscript!)\n\nEvaluation of antifungal activity: The writing style need serious improvements. This is not a laboratory report, the audience of this papers understand that the concentration of the substance and the inhibition halo are related. In the first paragraph, the authors refers the work with bacteria (?) , since the target microorganisms are fungi. The activation of lyophilized spores is routine lab work, please drop it from this section. Why was the concentration of the inoculum was not tested in the same way? The use of a Neubauer chamber allows to express the inoculum concentration in UFC as well. Expressing the “planting value” of the inoculum as 106 makes no sense. Then comes another situation: parts per million as concentration unit for plant extracts dilutions in an antimicrobial assay is never used. If the authors review the literature, they will note that the unit used is micrograms per milliliter (mg/mL). Please, use an space between the unit and the figure. DMSO is a solvent, not a diluent. The extracts and essential oils were placed, not planted, in the plate prepared for the bioassay. Please read more scientific papers regarding the agar well diffusion method to present a coherent methodology.\nIn the results, the authors are presenting tables copied directly from the software used to process the data. This is not admissible, even for undergraduate work. Besides, the antifungal activity of the extract and the essential oil are rather questionable. The highest antifungal activity of A. arborescens extract towards T. metagrophytes, T. rubrum and M canis, according to the results of this paper is 700 mg/mL. An antimicrobial activity for a plant extract is considered promising in concentrations below 100 mg/mL (Rios & Recio, 2005). Since the authors do not present the yield the essential oil extraction, is not possible to assess the feasibility of obtaining matico essential oil to scale the bioactivity in a pharmaceutical product. The results sections just limit to present the statistical analysis, without an evaluation of the real potential of the bioactivity of the plants studied.\nThe conclusions are a mere repetitions of the results of the laboratory work and statistical analysis.\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?\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": "38873", "date": "08 Oct 2018", "name": "Francesca Mancianti", "expertise": [ "Reviewer Expertise Mycology", "parasitology" ], "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 refers to the in vitro antifungal efficacy of both an ethanol extract from Ambrosia arborescens and Aristeguietia glutinosa essential oil. The topic is of interest, unfortunately the paper is not well written and deep editing by a native English speaker is needed. Furthermore references and data about these ethnobotanical species are too scanty. The composition of these products is not reported, making it difficult to reproduce the study. The authors should provide some data about the composition, related to healing ability, as well as the composition of specimens used for the present study.\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?\nYes\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/7-559
https://f1000research.com/articles/7-440/v1	10 Apr 18	{ "type": "Case Report", "title": "Case Report: First report of Elizabethkingia miricola infection in a patient with cystic fibrosis", "authors": [ "Freddy Frost", "Dilip Nazareth", "Dilip Nazareth" ], "abstract": "Elizabethkingia miricola is a rare non-fermenting Gram-negative rod that has previously been reported to be associated with blood stream and pulmonary abscess infections, but never before in cystic fibrosis (CF). Here we present the first reported case of Elizabethkingia miricola infection in a patient with CF and discuss the management options. We describe a patient with CF in whom we observed clinical and spirometric evidence of pulmonary exacerbation with the associated growth of E. miricola in sputum culture. The period of clinical instability was observed to coincide with the obtainment of four sputum samples from which E. miricola was cultured; improvement was seen following treatment with ciprofloxacin and the subsequent eradication of E. miricola. We conclude that E. miricola is able to survive in the CF lung and in this case was associated with pulmonary exacerbation. Empirical treatment with fluoroquinolones is appropriate, based on our experience.", "keywords": [ "Cystic fibrosis", "shortness of breath", "chest infection", "exacerbation", "fluoroquinolone" ], "content": "Introduction\n\nElizabethkingia miricola, a non-fermenting Gram-negative rod (NFGNB) was first identified following isolation from condensation water in the Russian space laboratory Mir1. Originally identified as belonging to the Chryseobacterium genus, it has since been re-classified and is closely related to Elizabethkingia. meningoseptica (previously C. meningosepticum). E. miricola has been demonstrated to be pathogenic, with reports of bacteraemia resulting in sepsis and pulmonary abscesses2,3. Here, we report the presence of E. miricola in the sputum of a patient with cystic fibrosis (CF). To our knowledge, this is the first reported case of E. miricola infection in CF; herein, we discuss the case itself and the literature surrounding this bacterium to help guide clinicians faced with similar clinical scenarios.\n\n\nCase report\n\nA 49-year-old male with a diagnosis of CF presented to his routine CF outpatient department complaining of feeling generally unwell. He reported increased cough, but this was non-productive. There was a drop in lung function, from a baseline forced expiratory volume in one second (FEV1) of 2.39 l (65% of the predicted volume) to 2.19 l (60% predicted). A sputum sample was taken and sent for routine culture, but given the non-specific symptoms and mild drop in FEV1, it was agreed that no immediate treatment was required and a follow-up in 4 weeks’ time was arranged.\n\nCo-morbidities of the patient included osteoporosis and pancreatic insufficiency; he was also receiving treatment for allergic bronchopulmonary aspergillosis (ABPA). Cultured respiratory samples in the previous year had consistently grown non-epidemic Pseudomonas aeruginosa. The diagnosis of CF was made in adulthood and was based the presence of bilateral bronchiectasis on a chest CT scan, a raised chloride level following a skin sweat test and genetic testing, which revealed that the patient was heterozygous for the CFTR F508del mutation. Family history included a younger sister who had died aged 23 years from pancreatitis.\n\nA sputum sample taken at the clinic appointment was positive for P. aeruginosa, and extended incubation isolated Elizabethkingia miricola. At the next appointment, worsening symptoms were observed, including increasing shortness of breath, wheeze and productive cough. There was a further drop in FEV1 to 1.91 L (52% predicted) (Figure 1). On the basis of outcomes of previous infective exacerbations, an oral course of chloramphenicol (500 mg four times a day) along with prednisolone (30 mg daily) for 2 weeks was commenced and a further sputum sample was obtained. Sputum culture was again positive for P. aeruginosa and E. miricola.\n\nLung function wsa measured using forced expiratory volume in one second (FEV1). Dotted lines represent the time period between which four sputum cultures were positive for E. miricola.\n\nA further 4 weeks later, symptoms were somewhat improved and FEV1 lung function had increased to 2.19 l (60% predicted). However, another 2 weeks later, symptoms deteriorated again, with an associated decline in lung function (FEV1, 1.95 l; 53% predicted). Sputum cultures from the previous admission were again positive for P. aeruginosa and E. miricola. Sensitivities from previous samples revealed E. miricola resistant to meropenem and ceftazidime, but sensitive to piperacillin/tazobactam and ciprofloxacin (CIP). A 2-week course of oral CIP (750 mg thrice daily) was therefore commenced.\n\nThe patient noted an improvement in symptoms and at the next clinic appointment FEV1 had improved to 2.08 l (57% predicted). Sputum then grew P. aeruginosa and yeast only. A further four subsequent sputum samples 1, 4, 8 and 12 months later have grown P. aeruginosa but no E. miricola, and lung function returned towards baseline.\n\n\nDiscussion\n\nE. miricola has been described in a number of healthcare settings, but not previously in CF. One of the first reports of E. miricola infection was of positive growth in blood and sputum cultures of a septic patient whom had recently undergone a stem-cell transplant for mantle-cell lymphoma. Since then it has been reported in only a handful of cases, including septicaemia in a young patient with alcoholic pancreatitis and in the sputum of a septic patient with pulmonary abscesses2,3. More recently, E. miricola was identified as causing a UTI in an immunocompetent adult4.\n\nHere, we describe a patient with CF in whom we observed clinical and spirometric evidence of pulmonary exacerbation, with associated growth of E. miricola in sputum culture. The period of clinical instability was observed to coincide with four sputum samples culturing E. miricola and improvement was seen with treatment. This is the first report of E. miricola in an individual with CF, meaning this report should therefore be relevant to all CF clinicians and microbiologists involved in the care of people with CF.\n\nAll case reports of E. miricola infection mentioned above report identification by MALDI-TOF (matrix-assisted laser desorption/ionisation time-of-flight) mass spectrometry2–4. MALDI-TOF has been widely adopted for bacterial identification, facilitating diagnosis quickly and reliably. In the CF setting, MALDI-TOF has also been shown to be particularly useful in identifying non-fermenting gram-negative bacteria, for which classification can be difficult using conventional phenotypic approaches5. Given the increasing use of MALDI-TOF in clinical microbiology laboratories, identification of NFGNB infections is likely to rise. Hence, establishing the optimal initial management strategies for these infections is important.\n\nIn this case, initial empirical treatment with oral chloramphenicol did not clear the infection, but treatment with oral CIP (based on culture sensitivities) successfully treated the exacerbation. Eradication of E. miricola was also observed, with contemporaneous clinical improvement. Notably, the sputum culture sensitivities revealed that P. aeruginosa was resistant to CIP, further supporting the idea that E. miricola had a pathogenic role. Our experience of treatment with a fluoroquinolone is in keeping with that of previous reports, in which E. miricola bacteraemia has been associated with sensitivity to levofloxacin and/or CIP, both of which resulted in successful treatment2,6,7. Susceptibility to co-trimoxazole (SXT) has also been reported, and it would seem that treatment with fluoroquinolones or SXT is an appropriate empirical strategy.\n\n\nConclusion\n\nE. miricola appears to have the potential to grow in the CF lung and can be associated with pulmonary exacerbation. Given the paucity of information on E. miricola infection in CF, we hope that the case report and literature review herein are relevant to CF clinicians and microbiologists alike. Treatment based on culture sensitivity is recommended, but empirical treatment with fluoroquinolones may be an appropriate initial strategy if there is suspicion of pathogenicity.\n\n\nConsent\n\nWritten informed consent for the creation and publication of this report was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "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\nLi Y, Kawamura Y, Fujiwara N, et al.: Chryseobacterium miricola sp. nov., a novel species isolated from condensation water of space station Mir. Syst Appl Microbiol. 2003; 26(4): 523–8. PubMed Abstract \| Publisher Full Text\n\nGreen O, Murray P, Gea-Banacloche JC: Sepsis caused by Elizabethkingia miricola successfully treated with tigecycline and levofloxacin. Diagn Microbiol Infect Dis. 2008; 62(4): 430–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonzalez C, Coolen-Allou N, Allyn J, et al.: [Severe sepsis and pulmonary abscess with bacteremia due to Elizabethkingia miricola]. Med Mal Infect. 2016; 46(1): 49–51. PubMed Abstract \| Publisher Full Text\n\nGupta P, Zaman K, Mohan B, et al.: Elizabethkingia miricola: A rare non-fermenter causing urinary tract infection. World J Clin Cases. 2017; 5(5): 187–190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlby K, Gilligan PH, Miller MB: Comparison of matrix-assisted laser desorption ionization-time of flight (maldi-tof) mass spectrometry platforms for the identification of gram-negative rods from patients with cystic fibrosis. J Clin Microbiol. 2013; 51(11): 3852–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRossati A, Kroumova V, Bargiacchi O, et al.: Elizabethkingia miricola bacteriemia in a young woman with acute alcoholic pancreatitis. Presse Med. 2015; 44(10): 1071–2. PubMed Abstract \| Publisher Full Text\n\nEriksen HB, Gumpert H, Faurholt CH, et al.: Determination of Elizabethkingia Diversity by MALDI-TOF Mass Spectrometry and Whole-Genome Sequencing. Emerg Infect Dis. 2017; 23(2): 320–323. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "33066", "date": "23 Apr 2018", "name": "Simon C. Langton Hewer", "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 agree that this appears to be the first report of this organism in CF and as such this is a relevant and important article and so should be published. The Case Report para 2 states the patient was heterozygous for CFTR mutation F508del but does not give the other mutation. Readers (including me) would be interested to know the second mutation so this should be stated.\nPara 4 states treatment of P aeruginosa and E miricola took place with oral chloramphenicol. This is an unusual choice as P. aeruginosa would usually be treated with ciprofloxacin or IV therapies and/or nebuliser amino glycoside. It would be helpful to know why chloramphenicol was chosen and whether any neb antibiotics were used as well (which is likely the case).\n\nThese details should be included to give a more complete picture of the case.\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? 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": "3610", "date": "08 May 2018", "name": "Freddy Frost", "role": "Author Response", "response": "We are very grateful to Dr Langton-Hewer for his review and comments. In response to Dr Langton-Hewer’s comments: Only one gene was identified despite extended testing. The diagnosis of CF was made based on one gene, raised sweat test, upper lobe bronchiectasis and family history. We have now included more detail with regard to this point. Oral chloramphenicol was chosen empirically based on previous response and the patients desire to avoid photosensitivity associated with ciprofloxacin. We have no included more detail in the manuscript. Nebulised therapy included Cayston and TOBI, these were continued throughout and we have now included this detail within the manuscript" } ] }, { "id": "33065", "date": "30 Apr 2018", "name": "Andrew M. 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 is the first case described as far as I am aware of Elizabethkingia mircola in a patient with cystic fibrosis.The authors describe isolation of Elizabethkingia mircola in association with clinical deterioration of a patient, and subsequent clinical improvement and eradication of the organism following quinolone therapy.\nThe report could be further improved by addressing the points below:\nThe authors state in the first paragraph that the patient had a cough that was unproductive, but in contrast also state that a sputum sample was obtained. The report states that the patient was receiving treatment for ABPA; it is important to know what exactly the treatment was and in particular if this could have caused immunosuppression and lead to increased susceptibility to unusual infections. The authors state the patient was heterozygous for F508del; did they identify the other CF gene? The authors state Elizabethkingia mircola was isolated after extended incubation: what culture media was used and how long was the extended incubation before Elizabethkingia mircola was isolated?\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? 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": "3626", "date": "08 May 2018", "name": "Freddy Frost", "role": "Author Response", "response": "We are grateful to Professor Jones for his time and review of our case-report. We have moved to clarify the points he has raised. In particular we have reviewed the electronic patient record and clarified that sputum sample was actually obtained by our physiotherapist. We have also expanded on the treatment for ABPA which did include low-dose oral corticosteroids and have discussed the relevance of this in our discussion. We have confirmed that only one gene was present despite extended genotype screening, but highlighted the reasons for making a robust clinical diagnosis of CF. We have also confirmed that growth was seen on the cepacia selective plate." } ] } ]	1	https://f1000research.com/articles/7-440
https://f1000research.com/articles/7-210/v1	21 Feb 18	{ "type": "Research Article", "title": "Knowledge discovery for Deep Phenotyping serious mental illness from Electronic Mental Health records", "authors": [ "Richard G Jackson", "Rashmi Patel", "Sumithra Velupillai", "George Gkotsis", "David Hoyle", "Robert Stewart", "Richard G Jackson", "Rashmi Patel", "Sumithra Velupillai", "George Gkotsis", "David Hoyle" ], "abstract": "Background: Deep Phenotyping is the precise and comprehensive analysis of phenotypic features, where the individual components of the phenotype are observed and described. In UK mental health clinical practice, most clinically relevant information is recorded as free text in the Electronic Health Record, and offers a granularity of information beyond that expressed in most medical knowledge bases. The SNOMED CT nomenclature potentially offers the means to model such information at scale, yet given a sufficiently large body of clinical text collected over many years, it’s difficult to identify the language that clinicians favour to express concepts. Methods: Vector space models of language seek to represent the relationship between words in a corpus in terms of cosine distance between a series of vectors. When utilising a large corpus of healthcare data and combined with appropriate clustering techniques and manual curation, we explore how such models can be used for discovering vocabulary relevant to the task of phenotyping Serious Mental Illness (SMI) with only a small amount of prior knowledge. Results: 20 403 n-grams were derived and curated via a two stage methodology. The list was reduced to 557 putative concepts based on eliminating redundant information content. These were then organised into 9 distinct categories pertaining to different aspects of psychiatric assessment. 235 (42%) concepts were found to be depictions of putative clinical significance. Of these, 53 (10%) were identified having novel synonymy with existing SNOMED CT concepts. 106 (19%) had no mapping to SNOMED CT. Conclusions: We demonstrate a scalable approach to discovering new depictions of SMI symptomatology based on real world clinical observation. Such approaches may offer the opportunity to consider broader manifestations of SMI symptomatology than is typically assessed via current diagnostic frameworks, and create the potential for enhancing nomenclatures such as SNOMED CT based on real world depictions.", "keywords": [ "word2vec", "natural language processing", "serious mental illness", "electronic health records", "schizophrenia" ], "content": "Introduction\n\nThe dramatic decrease in genetic sequencing costs, coupled with the growth of our understanding of the molecular basis of diseases, has led to the identification of increasingly granular subsets of disease populations that were once thought of as homogenous groups. As of 2010, the molecular basis for nearly 4 000 Mendelian disorders has been discovered1, subsequently leading to the development of around 2 000 clinical genetic tests2. The resulting ‘precision medicine’ paradigm has been touted as the logical evolution of evidence-based medicine.\n\nPrecision medicine has arisen in response to the fact that the ‘real world’ application of many treatments have a lower efficacy and a differential safety profile compared to clinical trials, most likely due to genetic and environmental differences in the disease population. Precision medicine seeks to obtain deeper genotypic and phenotypic knowledge of the disease population, in order to offer tailored care plans with evidence-based outcomes. Amongst the challenges presented by precision medicine is the requirement to obtain highly granular phenotypic knowledge that can adequately explain the variable manifestation of disease.\n\nTo realise the ambitions of precision medicine, large amounts of phenotypic data are required to provide sufficient statistical power in tightly defined patient cohorts (so called ‘Deep Phenotyping’3). Historical clinical data mined from Electronic Health Record (EHR) systems are frequently employed to meet the related use case of observational epidemiology. As such, EHRs are often posited as the means to provide extensive phenotypic information with a relatively low cost of collection4,5.\n\nIn order to standardise knowledge representation of clinically relevant entities and the relationships between them, phenotyping from EHRs often employ curated terminology systems, most commonly SNOMED CT. The use of such resources creates a common domain language in the clinical setting, theoretically allowing an unambiguous interpretation of events to be shared within and between healthcare organisations. The anticipated value of such a capability has prompted the UK National Information Board to recommend the adoption of SNOMED CT across all care settings by 20206. However, the task of representing the sprawling and ever-changing landscape of healthcare in such a fashion has proven complex7–10. Although a complete description of the structure and challenges of SNOMED CT are beyond the scope of this paper, we describe how aspects of these problems manifest themselves in accordance with the task of phenotyping serious mental illness (SMI) from a real world EHR system.\n\nThe quest for empirically validated criteria for assessing the symptomatology of mental illness has been a long term goal of evidence-based psychiatry. SMI is a commonly used umbrella term to denote the controversial diagnoses of schizophrenia (encoded in SNOMED as SCTID: 58214004), bipolar disorder (SCTID: 13746004), and schizoaffective disorder (SCTID: 68890003). While field trials of DSM-5 have revealed promising progress in reliably delineating these three conditions in clinical assessment11, such diagnostic entities continue to have low clinical utility12–14. Recent evidence from genome-wide association studies appears to suggest that such disorders share common genetic loci, further countering the argument that SMI can be classified into discrete, high level diagnostic units15. In terms of clinical practice, the presenting phenotype of SMI is usually the basis for treatment. This is often characterised by abnormalities in various mental processes, which are in turn categorised according to broad groupings of clinically observable behaviours. For instance, ‘positive symptoms’ refer to the presence of behaviours not seen in unaffected individuals, such as hallucinations, delusional thinking and disorganised speech. Conversely, ‘negative symptoms’, such as poverty of speech and social withdrawal refer to the absence of normal behaviours. Such symptomatology assessments are organised via an appropriate framework such as Postive and Negative Symptom Scale16 or Brief Negative Symptom Scale17. Accordingly, SNOMED CT includes coverage for many of these symptoms, generally within the ‘Behaviour finding’ branch (SCTID: 844005).\n\nA qualifying factor regarding the adoption of SNOMED amongst SMI specialists might therefore require that the list of clinical ‘finding’ entities in SNOMED are sufficiently expansive and diverse to represent their own experiences during patient interactions. Specifically, this may manifest as two key challenges for terminology developers:\n\nFirst, insight must be obtained regarding real-world language usage such that universally understood medical entities, encompassing hypernomy, synonymy and hyponomy adequately represent models of concepts. Similarly, the abundant use of acronyms in the medical domain has caused a large percentage of acronyms to have two or more meanings18, creating word sense disambiguation problems. As such, significant efforts have arisen to supplement these types of knowledge bases with appropriate real world synonym usage extracted from EHR datasets19.\n\nSecond, if there is controversy over international consensus in a particular area of medicine, the use of ‘global’ perspectives may not be sufficient to meet local reporting/investigatory requirements. Such issues are particularly pertinent in mental health where many diseases defy precise definition and biomarker development has yielded few successes20. More generally, all medical knowledge bases are incomplete to one degree or another. The opportunity to utilise large amounts of EHR data to discover novel observations and relationships arising from real world clinical practise must not be overlooked.\n\nGiven a sufficiently large corpus of documents, typically authored by hundreds of clinical staff over several years, it is often difficult to predict the diversity of vocabulary used within the local EHR setting to describe potentially important clinical constructs. In previous work, we describe our attempts to extract fifty well known SMI symptomatology concepts from a large electronic mental health database resource21, based upon the contents of such frameworks. During the course of manually reviewing clinical text, we made two subjective observations regarding the authorship of clinician/patient interactions:\n\nThe tendency of clinicians to use non-technical vocabulary in describing their observations\n\nThe occasional appearance of highly detailed, novel observations that do not readily fit into known symptomatology frameworks\n\nSuch observations may feasibly have clinical relevance, for example, as non-specific symptomatology prodromes22. On the basis that the modelling of SMI for precision medicine approaches require the full dimensionality of the disease to be considered, we sought to explore these observations further.\n\nIn this study, we present our efforts to utilise a priori knowledge discovery methods to identify patterns of real world language usage that reflect clinically relevant SMI symptomatology within the context of a large mental healthcare provider. We contrast and compare these patterns with a modern version of the UK SNOMED CT (v1.33.2), and suggest how such approaches may offer novel and/or more granular symptom depictions from patient/clinician interactions when used to supplement resources such as SNOMED CT, potentially offering alternatives to classify psychiatric disorders with finer resolution and greater real-world validity.\n\n\nMethods\n\nOur general approach for SMI knowledge discovery is composed of several discrete steps. An overview of the workflow is given in Figure 1.\n\nThe South London and Maudsley NHS Foundation Trust (SLaM) provides mental health services to 1.2 million residents over four south London boroughs (Lambeth, Southwark, Lewisham and Croydon). Since 2007, the Clinical Record Interactive Search (CRIS)23 infrastructure programme has been operating to offer a pseudonymised and de-identified research database of SLaM’s EHR system. As the CRIS resource received ethical approval as a pseudonymised and de-identified data source by Oxford Research Ethics Committee (reference 08/H0606/71+5), patient consent was not required for this study.\n\n11 745 094 clinical documents were collected from the CRIS database from the period 01/01/2007 - 27/10/2016 on the basis that the 20 472 associated patients were assigned an SMI ICD10 code of F20, F25, F30 or F31 at some point during their care, in accordance with current clinical practice.\n\nSentences and tokens were extracted from each document using the English Punkt tokeniser from the NLTK 3.0 suite24. Each token was converted to lower case. A vocabulary was then constructed of all 1-gram types in the corpus, supplemented with frequently occuring bi-grams and tri-grams using the Gensim25 suite and the sampling method proposed by Mikolov et al.26. Bi-grams and tri-grams with a minimum frequency of 10 occurrences in the entire corpus were retained, to give a total vocabulary size of 896 195 n-grams. No further assumptions about the structure of the data, such as the need for stemming/lemmatisation, were made.\n\nThe distributional hypothesis was first explored by Harris27, which proposed that, given a sufficiently large body of text, linguistic units that co-occur in the same context are likely to have a semantically related meaning. Modelling the distribution of such units may therefore have value for a wide range of natural language processing applications. Models of distributional semantics, including word embeddings, are techniques that aim to derive models of semantically similar units in a corpus of text by co-locating them in vector space. In recent years, the use of the Continuous Bag-of-Words (CBOW) model proposed by Mikolov et al.28 has risen to prominence, owing to its ability to accurately capture semantic relationships whilst scaling to large corpora of text26. Recently, the CBOW model has been used to identify the semantic similarities between single word entities in biomedical literature and clinical text29, suggesting that biomedical literature may serve as a useful proxy for clinical text, for tasks such as synonym identification and word sense disambiguation tasks under limited conditions29.\n\nA full description of the CBOW architecture is discussed in 30. For brevity, we describe only the key features used in our work here. The purpose of the architecture is to ’learn’ in an unsupervised manner, a representation of the semantics of different n-grams, given an input set of documents. CBOW might be described as a simple feed forward neural network consisting of three layers. An input layer X composed of o nodes (where o is the number of unique n-grams in a corpus produced from our above described pre-processing), a hidden layer H of a user defined size n (usually between 100 and 300), and an output layer Y that is also composed of o nodes. Every node in X is connected to every node in H, and every node in H is connected to every node in Y. Between each of the layers is a matrix of weight values; for the X and H layer, an ‘input’ matrix of dimensions o × n (hereafter denoted W); and between the H and the Y layer, an ‘output’ matrix of dimensions n × o (denoted W′). The output of training the neural network is to produce weights in each of these matrices. The weights learnt in the W matrix might be intuitively described as the semantic relationships between each n-gram in the vocabulary as represented in vector space, with semantically similar words located in closer proximity to each other. Weights in the W′ matrix represent the predictive model from the H to the Y layer. A training instance is composed of a group of n-grams, known as a context. A context can be composed of natural language structures, such as sentences in a document, or more complex arrangements, such as a sliding window of n-grams (usually between 5 and 10) that move over each token in a document (potentially ignoring natural grammatical structures). For a given input n-gram, the input into the nodes on the hidden layer is the product of each vector index in matrix W corresponding to each context word and the average vector. From the H to the Y layer, it is then possible to score each n-gram using the W′ matrix, from which a posterior probability is obtained for each word in the vocabulary using the softmax function. The weights in each matrix are then updated using computationally efficient hierarchical softmax or negative sampling approaches. Once training is complete, the semantic similarity of n-grams is often measured via their cosine distance between vectors in the W matrix.\n\nUsing the Gensim implementation of CBOW and our previously constructed vocabulary, we trained a word embedding model of n = 100 over our SMI corpus to produce a vector space representation of our clinical vocabulary. Due to patient confidentiality, offline access to records was not feasible and so only a limited number of epochs of training could be performed. However, due to the relatively narrow/controlled vocabulary employed in clinical records (compared to normal speech/text) the range of possible input vectors was narrower than might otherwise be expected, and even a single epoch of training appeared to yield meaningful clusters that could be identified with SMI. As we were primarily intending to identify initial clusters for validation by clinical experts it was felt that single epoch of training, over the 20M clinical records available, was sufficient.\n\nThe task of clustering seeks to group similar dataset objects together in meaningful ways. In unsupervised clustering, the definition of ‘meaningfulness’ is often subjectively defined by the human observers. In our task, we sought to identify clusters of n-grams derived from our word embedding model that represent semantically linked components of our clinical vocabulary, based on the theory that our word embedding model would cause related symptom concepts to appear close to each other within the vector space.\n\nA particular challenge in the development of clustering algorithms is achieving scalability to large datasets. Since many clustering algorithms make use of the pairwise distance between n samples (or n-grams, in our case), the memory requirements of such algorithms tend to run in the order of n2. One such algorithm that does not suffer from this limitation is k-means clustering. k-means clustering is a partitional clustering algorithm that seeks to assign n samples into a user defined k clusters by minimising the squared error between each centroid of a cluster and its surrounding points. A global (although not necessarily optimal) solution is derived when the algorithm has minimised the sum of squared errors across all k clusters, subject to some improvement threshold or other stopping criteria. For all experiments, we used the k-means++ implementation from the Scikit-Learn framework31 with 8 runs each time, to control against centroids emerging in local minima.\n\nThe key parameter for k-means clustering is the selection of k. While techniques exist for estimating an appropriate value, such as silhouette analysis and the ‘elbow method’32, these utilise pairwise distances between samples, creating substantial technical limitations for large matrices in terms of memory usage. To overcome this, we opted for a memory efficient version of the elbow method, involving plotting the minimum centroid distance for different values of k. The intuition behind this approach is that every increase in k is likely to result in a smaller minimum centroid distance in vector space (subject to a random seed for the algorithm). As k increases, genuine clusters should be separated by a steady decline in minimum centroid distance. However, when the slope of the decline flattens out (i.e. the ‘elbow’ of the curve), assignment of samples to new clusters is likely to be random).\n\nTo identify a putative cluster of SMI symptomatology, we devised a simple ‘relevance’ cluster scoring approach based upon prior knowledge of common SMI symptom concepts. We selected 38 symptoms of SMI based upon their depictions in SMI frameworks and on their specificity in clinical use (Table 1. For instance, we did not select ‘loosening of associations’, due to the different word sense that the word ‘associations’ appears in, such as ‘housing associations’, and organisational references such as ‘Stroke Association’. Rather, we chose symptoms such as ‘aggression’, ‘apathy’ and ‘agitation’, which are less likely to have different word sense interpretations in the context of SMI clinical documents. We produced stems, appropriate synonyms/acronyms and/or n-grams of each concept as described in Table 1. With this matching criterion, we scored each cluster based on the number of per concept hits to derive a cluster/concept count matrix x where xi,j represents the count of the ith concept in the jth cluster. For example, a cluster containing the terms ‘insomnia’ and ‘insomniac’ would receive a count of two for the ‘insomni’ concept. For each concept, we then calculated a vector of the minimum count per concept across all clusters:\n\n\n\nAn underscore represents a bigram match.\n\nto enable us to rescale the value of each concept/cluster count to between 0 and 1 into a matrix x′:\n\n\n\nThe purpose of rescaling in such a way was to prevent overrepresented concepts unduly influencing the overall result (for instance, a concept with many hits in a cluster would unduly bias the score towards that concept, whereas we sought a scoring mechanism that would weigh all input concepts equally, regardless of their frequency).\n\nFinally, we summed all rescaled concept counts per cluster, and divided by the total cluster size to provide a score per cluster z representing known symptomatology contents:\n\n\n\nwhere s is a vector of the total count of n-grams in each cluster. The purpose of dividing by cluster size was to prevent the tendency of larger clusters to score higher on account of their size.\n\nTo select clusters for further investigation, the robust median absolute deviation (MAD) statistic was chosen to identify outliers (the distribution of our cluster scores was non-normal). We adopted a conservative approach to cluster selection by choosing clusters that scored at least six MAD above the median score for further processing, which is approximately equivalent to four standard deviations for a normally distributed dataset.\n\nThe contents of the top scoring clusters underwent a two stage curation process. The first stage was performed by an informatician, and involved a simple manual data cleaning process to reduce the total number of concepts via grouping misspellings, tokenisation failures and other constructs that did not yield additional information content.\n\nThe second, more important stage was composed of independent annotation of the curated concept list by two psychiatrists, to identify likely synonyms and new symptomatology based on their clinical experience. Each concept was assigned to one of the below 8 ‘substantive’ categories, or a 9th ‘other’ category.\n\nAppearance/Behaviour Implying a real-time description of the way a patient appears or behaves (including their interaction with the recording clinician)\n\nSpeech Anything implying a description of any vocalisation (i.e. theoretically a subset of behaviour but restricted to vocalisations)\n\nAffect/Mood Implying clinician-observed mood/emotional state (i.e. theoretically a subset of appearance but restricted to observed emotion), or implying self-reported mood/emotional state (i.e. has to imply a description that a patient would make of their own mood; theoretically a subset of thought)\n\nThought Implying any other thought content\n\nPerception Implying any described perception\n\nCognition Implying anything relating to the patient’s cognitive function\n\nInsight Implying anything relating to insight (awareness of health state)\n\nPersonality Anything implying a personality trait or attitude (i.e. something more long-standing than an observed behaviour at interview)\n\nOther A mixed bag of definable terms that do not fit into the above. Common examples included anything implying information that will have been collected as part of a patient’s history, often of behaviours that would have to have been reported as occurring in the past and cannot have been observed at interview, but also which cannot be termed a personality trait. Alternatively, anything where insufficient context was available to make a decision\n\nInter annotator agreement (IAA) was measured with the Cohen’s Kappa agreement statistic33.\n\nTo explore the frequency of both our prior symptomatology concepts and the newly curated ones in our symptom clusters, we counted the number of unique patient records and the number of unique documents that the stems of each n-gram appeared in. To protect patient anonymity, we discarded any concept that appeared in ten or less unique patient records. Finally, we mapped the remaining concepts to SNOMED CT, UK version v1.33.2, using the following method. First, the root mopheme of each concept was matched to a relevant finding, observable entity or disorder type in SNOMED CT. If a match could not be found, SNOMED CT was explored for potential synonymy, or other partial match. If a clear synonym could not be found, we classified the concept as novel.\n\n\nResults\n\nProcessing the corpus of SMI clinical documents took approximately 100 hours on an 8-core commodity hardware server. Documents were fed sequentially from an SQL Server 2008 database operating as a shared resource, with additional overhead likely resulting from network latency.\n\nFigure 2 shows a scatterplot of variable values of k and the resulting minimum centroid distance. This suggests a k value of around 50-75 may be optimal for our data. On this basis, we chose a k value of 75.\n\nThe elbow of the curve indicates the point at which clearly defined clusters no longer emerge with higher values of K.\n\nThe application of our relevancy scoring algorithm to the 75 derived clusters resulted in a median score was 0.000229 and a MAD of 0.000277, and is visualised in Figure 3.\n\nEach dot represents a unique cluster. The unique cluster ID’s of the most relevant clusters according to our scoring algorithm are labelled.\n\nThree clusters emerged with a score at least six MADs outside of the median cluster score: No. 52 (score: 0.002883), containing 6 665 terms, No. 69, containing 9 314 (score: 0.002282) terms and No. 49 (score: 0.001940), containing 4 424 terms. Taken together, these three clusters contained a total of 20 403 n-grams.\n\nThe combined 20 403 n-grams were taken forward for curation as described above. The first phase of curation reduced the list to 519 putative concepts. The majority of eliminated concepts were morphological variations, misspellings and tokenisation anomalies of singular concepts. For instance, 84 variations were detected for the stem ‘irrit*’ (as in ‘irritable’). Other n-grams were removed because insufficient context was available for a reasonable clinical interpretation, such as ‘fundamentally unchanged’, ’amusing’ and ‘formally tested’. Finally, n-grams that appeared to have no relevance to symptomatology at all were removed, such as dates and clinician names.\n\nExpert curation by two psychiatrists of the 557 terms (519 discovered concepts and 38 prior concepts) produced a Cohen’s Kappa agreement score of 0.45, where 337 concepts were assigned to one of our 9 categories independently by expert psychiatric curation. Of the 337 concepts, 235 were assigned to a substantive category (i.e. not the indeterminate ‘other’ group). Table 2 shows the number of n-grams per category where agreement was reached.\n\nSupplementary File 1 is a CSV table of all 557 n-grams. In addition to the n-gram itself, the table contains the following information; the counts of the unique patient records of our 20 472 patient SMI cohort in which the n-gram was detected; the counts of the unique documents of the 11 745 094 clinical document corpus wherein the n-gram was detected; the category assigned to the n-gram by each of our clinical annotators, and the SNOMED CT ID code for each n-gram, where mapping was possible.\n\nThe most frequently detected concept mentions include ‘affect’ (detected in 91% of patients), ‘eye contact’ (85%), ‘hallucinations’ (85%), ’delusions’ (83%) and ‘rapport’ (81%). Other concepts follow a long tailed distribution, with mentions of the top 407 concepts found in at least 100 unique patient records.\n\nRegarding SNOMED CT mapping, it was possible to suggest direct mappings for 177 concepts and to suggest synonymy or partial mapping for another 53 concepts. This left a remaining 327 concepts that did not appear to be referenced in SNOMED CT, of which 106 were classified as belonging to a substantive symptom category by independent curation.\n\nFigure 4 visualises the top 20% most frequent n-grams by appearance in unique patient records, where annotators agreed and were not classified as our ‘other’ grouping. Owing to the difficulty of the IAA and categorisation task, an extended analysis of the top 40% most frequent n-grams by appearance in unique patient records, irrespective of IAA and categorisation is provided in Supplementary Figure 1.\n\nWhite bars represent concepts that were found to exist in SNOMED CT. Grey bars represent partial/uncertain matches, or novel synonyms of existing SNOMED CT concepts. Black bars represent concepts with no SNOMED mapping.\n\nIn this project, we sought to explore SMI symptomatology and other language constructs as expressed by clinicians in their own words, using more than ten years of observations made during real world clinician/patient interactions from more than 20 000 unique SMI cases. Within the context of a large mental healthcare provider, the results of our vocabulary curation efforts suggest that psychiatrists make use of a wide range of vocabulary to describe detailed symptomatic observations.\n\nMany of the curated entities where both annotators agreed upon a substantive category map directly to preferred terms or synonyms of well known symptomatology constructs as described in SNOMED CT. Reassuringly, many of most frequently encountered entities as represented by unique patient count are represented in SNOMED CT, suggesting that SNOMED CT offers a reasonable coverage of what clinicians deem to be the most salient features of a psychiatric examination.\n\nNevertheless, our work produces evidence to suggest that many suitable synonyms are currently missing from SNOMED CT symptom entities. For instance, ‘aggression’ is commonly observed in SMI patients. Our results indicate that this construct might also be referred to by adjectives and phrases such as ‘combatative’ [sic], ‘assaultative’ [sic], ‘truculent’, ‘stared intimidatingly’ and ‘stared menacingly’, amongst others. Similarly, direct synonyms of ‘paranoia’ might include ‘suspiciousness’, ‘mistrustful’ and ‘conspirational’[sic].\n\nIn addition, many of the curated constructs appear to reflect more granular observations of known symptomatology. For example, the Positive and Negative Symptom Scale (PANSS) utilises a 30 point scale of different symptomatology constructs. Specifically regarding abnormal speech, the PANSS provide guidance amounting to the high level clinical scrutiny of ‘lack of spontaneity & flow of conversation’. However, clinical depictions of speech within our dataset suggest around 68 distinct states, including ‘making animal noises’, ‘staccato quality’, ‘easily interruptible’, ‘prosody’ and ‘silently mouthing’.\n\nWe note the occurrence of several constructs that defy classification under existing schemas of SMI symptomatology, such as behaviours of ‘over politeness’, ‘over complimentary’, ‘spending recklessly’ and ‘shadow boxing’. The clinical interpretation of such entities is a non-trivial exercise, and is out of scope for this piece. Nevertheless, word embedding models may offer the potential to gain insight into potentially novel symptomatology constructs observed from real world clinician/patient interactions. Future work might explore the context for such constructs in more detail.\n\nThe emergence of such diverse language in turn has implications for how SNOMED CT might be implemented within an SMI context, raising the question of whether such gaps represent significant barriers to the use of SNOMED CT as a phenotyping resource. The issue of SNOMED CT’s sufficiency in this context has previously been raised for other areas, such as rare disease34, psychological assessment instruments35 and histopathology findings36. However, in fairness, SNOMED CT is not a static resource, but an international effort dependent on the contributions of researchers. Perhaps a more pertinent question for the future development of SNOMED CT concerns balancing its objective to be a comprehensive terminology of clinical language (capable of facilitating interoperability and modelling deep phenotypes within disparate healthcare organisations across the globe) and the overwhelming complexity it would need to encompass in order to not constain its users. Certainly, at over 300 000 entities in its current incarnation, its size already presents problems in biomedical applications37.\n\nOn the basis that manifestations of symptoms are the result of abnormal mental processes, novel symptom entities possibly represent observations of clinical significance. However, one particular complication in validating the clinical utility of novel symptomatology constructs with historic routinely recorded notes arises from systemic biases in EHR data. Specifically, the breadth and depth of symptomatic reporting is likely to be highly variable for a number of reasons. For instance, established symptoms as defined by current diagnostic frameworks are likely to be preferentially recorded, as clinicians are mandated to capture such entities in their assessments. On the other hand, constructs that fall outside of such frameworks may only be recorded as tangential observations made during patient/clinician interactions. Regardless of whether they are observed or not, without an established precedent of their clinical utility, they may be subject to random variation as to whether they are documented in a patient’s notes. This is borne out by the tendency of SNOMED CT-ratified concepts to appear more frequently in unique documents compared to our derived expressions. The validation of new symptoms from historic data is therefore something of a ‘chicken and the egg’ situation, a widely discussed limitation of the reuse of EHR data38,39. Nevertheless, our frequency analysis of our discovered constructs suggests that there is evidence that many are observed often enough to warrant their consideration within an expanded framework. Similarly, older frameworks with a limited scope of symptomatic expression were likely designed with pragmatic constraints around speed and reproducibility of assessment in mind. However, modern technology allows for a far greater scope of data capture and validation going forward, creating opportunities to develop new frameworks that maximise the value of psychiatric assessment. Future work in this domain might seek statistical validation via randomised experimental design, as opposed to observational study.\n\nOur work suggests an approximate correlation between patient and document count, such that intra and inter patient symptomatological clinical language usage varies relatively consistently. However, some notable exceptions to this correlation (i.e. with a higher document level frequency to patient record level frequency) include ‘aggression’, ‘pacing’, ‘sexual inappropriateness’, ‘sexual disinhibition’ and ‘mutism’. Further work might seek to study these effects in greater detail, to uncover whether they represent a systemic bias in how such concepts are represented in the EHR.\n\nThe results of our IAA exercise between two experienced psychiatrists suggested a moderate level of agreement in categorising the newly identified constructs. Given that this annotation exercise did not provide any context beyond the n-gram, and that the nature of SMI symptom observation is somewhat subjective, perhaps it is to be expected that agreement was not higher. Nevertheless, future work might seek more formal definitions of such constructs via expert panel discussion and engagement with international collaborative efforts in SMI research.\n\nOur method for vocabulary building produced nearly 1 million n-grams. A manual annotation of this list may have resulted in further discoveries, although would have been intractable in practical terms. To reduce the volume of n-grams taken forward for curation, we employed a word embedding model with a clustering algorithm. With our cluster scoring methodology, we were able to successfully produce meaningful clusters of n-grams reflecting the semantics of SMI symptomatology. However, as with many unsupervised tasks, it is difficult to determine whether an optimal solution has been achieved. In particular, the emergence of three ‘symptom’ clusters instead of one indicates sub-optimal localisation of symptom constructs in vector space. Addressing such a problem is multifaceted. For technical reasons, only a single epoch of training was possible in this exercise. Additional epochs would likely contribute to better cluster definition, in turn allowing us to reduce the value of our k parameter. In addition, spell checking and collapsing n-grams into their root forms may also have assisted. However, the latter may have also created new word sense disambiguation problems if common, symptom-like morphemes also appear in non-symptomatological assessment contexts.\n\nAfter clustering, a two stage manual curation of more than 20 000 n-grams was necessary. Methods that produce a smaller vocabulary might conceivably reduce annotator burden. This might include the use of spell checkers and stemming/lemmatisation to correct and normalise tokens, at the risk of introducing new issues associated with morphological forms in word embedding model building. For this attempt, we took the conscious decision to make as few assumptions about the underlying structure of the data as possible.\n\n\nConclusions\n\nEvidence-based mental health has long sought to produce disease model definitions that are both valid, in the sense they represent useful clinical representations that can inform treatment, and reliable, in that they can be consistently applied by different clinicians to achieve the same outcomes. In practice this has proven difficult, due to the often subjective nature of psychiatric examination/phenotyping and insufficient knowledge about the underlying mechanisms of disorders such as SMI. Here, we demonstrate that clinical staff make use of a diverse vocabulary in the course of their interactions with patients. This vocabulary often references findings that are not represented in SNOMED CT, raising questions about whether clinicians should observe the constraints of SNOMED CT or whether SNOMED CT should incorporate greater flexibility to reflect the nature of mental health. It is outside the scope of this work to explore how the granularity of symptom-based phenotyping affects patient outcomes, although the possibility of offering a fully realised picture of symptom manifestation may prove valuable in future endeavours of precision medicine.\n\n\nData availability\n\nThe CRIS dataset is a pseydonymised and de-identified case registrar of electronic health records of the South London and Maudsley NHS Trust. It operates under a security model that does not allow for open publication of raw data. However, access can be granted for research use cases under a patient led security model. For further information and details on the application process, please contact cris.administrator@kcl.ac.uk or visit the website. Alternatively, you may write to the CRIS team at: PO Box 92 Institute of Psychiatry, Psychology & Neuroscience at King’s College London 16 De Crespigny Park London SE5 8AF", "appendix": "Competing interests\n\n\n\nRJ and RS have received research funding from Roche, Pfizer, J&J and Lundbeck.\n\n\nGrant information\n\nThis paper represents independent research funded by the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s College London. RP has received support from a Medical Research Council (MRC) Health Data Research UK Fellowship and a Starter Grant for Clinical Lecturers (SGL015/1020) supported by the Academy of Medical Sciences, The Wellcome Trust, MRC, British Heart Foundation, Arthritis Research UK, the Royal College of Physicians and Diabetes UK. SV is supported by the Swedish Research Council (2015-00359), Marie Sklodowska Curie Actions, Cofund, Project INCA 600398.\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: This file contains all of the 557 terms taken forward for expert annotation. It includes SNOMED mappings where possible, unique document and patient counts within the corpus, and the annotations provided by RP and RS.\n\nClick here to access the data.\n\nSupplementary Figure 1: This file is an expanded visualisation of the frequency analysis figure contained in the main manuscript, with the agreement and non-substantive ‘other’ classification restrictions lifted.\n\nClick here to access the data.\n\n\nReferences\n\nAmberger J, Bocchini C, Hamosh A: A new face and new challenges for Online Mendelian Inheritance in Man (OMIM®). Hum Mutat. 2011; 32(5): 564–567. ISSN 10597794. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "31101", "date": "21 Mar 2018", "name": "Julian Hong", "expertise": [ "Reviewer Expertise Patient stratification in mental health using EHR data", "structured and free text." ], "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 method to extract from a clinical corpus novel terms used to described serious mental illness (SMI). They use vector space models to represent the relationship between words in the corpus and combine this approach with clustering techniques and manual curation to identify relevant n-grams (1, 2, or 3-word concepts). 106 concepts had no mapping to current SNOMED terms indicating that they have indeed discovered new knowledge, i.e. terms used by clinicians to describe patients that are not already included in SNOMED CT.\n\nThe introduction was unusually well written, if a little longer than strictly necessary, and provided excellent motivation for the work at hand. It has been shown that SNOMED coverage of mental health terms is sub-optimal and this is a clever approach to learning new relevant terms in a semi-automated manner. For the rest of the paper, each individual part was well written, but I had a hard time seeing how they flowed together. Figure 1 was a helpful overview, but I still found it difficult to follow how the sub-steps tied in together and in some cases why they were important. e.g.\nHow did creation of the putative cluster of 38 terms help? I think it was to facilitate the scoring method, but I wasn't completely clear how. Why 38? Particularly when the clusters they later looked at were so much larger, not clear why that number was chosen.\n\nI found the math/logic challenging to follow. (Admittedly, I am not a statistician, and was not previously familiar with CBOW.)\nIt was helpful that the authors included examples in some places, but they could have gone even further to make the approach concrete. Toy examples of u1 and v1 would help. On first and second read, I was having a hard time with intuition for what a high-scoring cluster means. I now realize (I think?) it meant the cluster was particularly enriched for mental health terms. It might be helpful to state that- for some reason I was thinking it meant that the concepts were relatively similar/cohesive? I had trouble wrapping my head around the sentence \"we scored each cluster based on the number of per concept hits to derive a cluster/concept count matrix x where xi,j represents the count of the ith concept in the jth cluster.\" I think it means 38 rows, 1 for each concept and 3 columns, 1 for each cluster, and the value of the cell is the number of times that concept was encountered in some form in the cluster? Equations could also be numbered for reference.\n\nThe authors report choosing not to perform stemming/lemmatization in order not to make assumptions about the structure of the data, but this decision is not very well explained or justified. Indeed they call it out as a potential limitation in Discussion. It would be useful/interesting to try the approach both ways and see if the results were different. How were the 8 \"substantive categories\" chosen? Why does inter-rater agreement matter in mapping the concepts to those categories? Was it only that the ability to map them to a single category makes it more likely the concept is semantically interesting and reliable? The authors mention that the semantic similarity of n-grams is often measured via their cosine distance between vectors in the W matrix. Just out of curiosity, could distance in the W' matrix be used as well/instead? My \"partly\" answer to \"Are sufficient details of methods and analysis provided to allow replication by others?\" reflects the fact that the authors very reasonably cannot publish the raw data, but they do address how to obtain the data through a formal application process. (Ergo the \"Yes\" to whether source data are available, even if not readily…) It would be helpful if code were to be made available.\n\nMinor:\nPage 3, paragraph 4 should be employS curated terminology Page 6 line 5 should have ) after \"Table 1\"\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3606", "date": "08 May 2018", "name": "Richard Jackson", "role": "Author Response", "response": "Thank you for your valuable comments. Please see our responses below For the rest of the paper, each individual part was well written, but I had a hard time seeing how they flowed together. Figure 1 was a helpful overview, but I still found it difficult to follow how the sub-steps tied in together and in some cases why they were important. e.g. How did creation of the putative cluster of 38 terms help? I think it was to facilitate the scoring method, but I wasn't completely clear how. Why 38? Particularly when the clusters they later looked at were so much larger, not clear why that number was chosen. We think there’s some misunderstanding of our methodology and apologise if it wasn’t clear in the manuscript. We didn’t create any clusters in this work by hand. All of the 896 195 terms generated from the corpus were assigned to one of 75 distinct clusters via the K-means algorithm. We then needed to identify which of the 75 clusters were worth looking at. The 38 concepts constitute the prior knowledge about SMI symptomatology that our clinical team fed into the scoring algorithm we describe in the manuscript. This revealed three clusters that we took forward for further analysis. We’ve re-written the text concerning this, and introduced something we call ‘Prior Concepts’ to differentiate between the domain knowledge we use in cluster scoring and the clusters themselves. Please do let us know if you feel that this hasn't improved the manuscript clarity. I found the math/logic challenging to follow. (Admittedly, I am not a statistician, and was not previously familiar with CBOW.) It was helpful that the authors included examples in some places, but they could have gone even further to make the approach concrete. Toy examples of u1 and v1 would help. We’ve added an example of the analysis pipeline to the accompanying code repository, and added the line: “ We provide a worked example of this technique in the code repository that accompanies this paper, using publically available data. “ On first and second read, I was having a hard time with intuition for what a high-scoring cluster means. I now realize (I think?) it meant the cluster was particularly enriched for mental health terms. It might be helpful to state that- for some reason I was thinking it meant that the concepts were relatively similar/cohesive? Actually you are correct on both counts. Training the Word2Vec model causes n-grams with semantically similar meanings to co-locate near to each other in the vector space model. The application of the clustering algorithm groups semantically similar n-grams together. Three clusters scored highly in our relevancy scoring algorithm, signifying that they were enriched for our existing knowledge of SMI symptomatology. I had trouble wrapping my head around the sentence \"we scored each cluster based on the number of per concept hits to derive a cluster/concept count matrix x where xi,j represents the count of the ith concept in the jth cluster.\" I think it means 38 rows, 1 for each concept and 3 columns, 1 for each cluster, and the value of the cell is the number of times that concept was encountered in some form in the cluster? This is almost correct, although j represents the total number of clusters (75). The result is a score per cluster that is plotted in figure 3. We have rewritten this section of text accordingly, as per the previous comment on this issue Equations could also be numbered for reference. This is now done The authors report choosing not to perform stemming/lemmatization in order not to make assumptions about the structure of the data, but this decision is not very well explained or justified. Indeed they call it out as a potential limitation in Discussion. It would be useful/interesting to try the approach both ways and see if the results were different. This is a potential limitation of our work, in that using un-stemmed tokens will have led to vastly more n-grams than we might have otherwise had to deal with, and that stemming might have lead to the identification of additional n-grams of interest. However, we feel our decision not to make assumptions about the value of stemming in this context was appropriate for two reasons: In the context of a mental health assessment, stemming may cause important information loss. For instance, the term ‘insomnia’ shares the same stem as ‘insomniac’. However, short term ‘insomnia’ is a relatively common symptom amongst the general population for a large variety of conditions. ‘Insomniac’ in the context of mental illness, on the other hand, might imply a chronic condition. Our IAA task would have been substantially more complex if we were to offer stemmed n-grams for human evaluation, rather than complete words. How were the 8 \"substantive categories\" chosen? The categories were derived from the Shorter Oxford Textbook of Psychiatry (chapter 3, page 44), and the experience of the teams Clinical Psychiatrists. We have updated the text as follows: “ The second, more important stage was composed of independent annotation of the curated concept list by two psychiatrists, to identify likely synonyms and new symptomatology based on their clinical experience. Each concept was assigned to one of the below 8 `substantive' categories, or a 9th `other' category. The categories were derived from\\cite{harrison_shorter_2018}, and the experience of the team Clinical Psychiatrists. ” Why does inter-rater agreement matter in mapping the concepts to those categories? Was it only that the ability to map them to a single category makes it more likely the concept is semantically interesting and reliable? Yes, we felt that offering a binary choice per n-gram (i.e. potentially relevant/irrelevant) was likely to heavily bias our results in favour of high agreement. Rather, we thought that agreement on the mapping of the identified n-grams to defined groups of symptomatology would suggest a greater degree of robustness. The authors mention that the semantic similarity of n-grams is often measured via their cosine distance between vectors in the W matrix. Just out of curiosity, could distance in the W' matrix be used as well/instead? We don’t believe this is possible, as the W matrix corresponds to weights between the input layer and the hidden layer of the neural network, where each row represents a single n-gram. The W' corresponds to the weights between the hidden layer and the output layer, which has different dimensions. I recommend this reference for a detailed description of the Word2Vec methodology. My \"partly\" answer to \"Are sufficient details of methods and analysis provided to allow replication by others?\" reflects the fact that the authors very reasonably cannot publish the raw data, but they do address how to obtain the data through a formal application process. (Ergo the \"Yes\" to whether source data are available, even if not readily…) It would be helpful if code were to be made available. We agree this would be useful, and now provide a link to a repository in the paper: “ Example code used in this analysis is available at: https://github.com/RichJackson/clustering_w2v ” Minor: Page 3, paragraph 4 should be employS curated terminology Page 6 line 5 should have ) after \"Table 1 This is now corrected. Thanks once again for your time and efforts with our paper. Best wishes Richard" } ] }, { "id": "31100", "date": "22 Mar 2018", "name": "Karin Verspoor", "expertise": [ "Reviewer Expertise biomedical natural language processing" ], "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 introduces a strategy for unsupervised analysis of a corpus of documents in order to identify terminology related to Serious Mental Illness (SMI). It applies a process of (1) identification of frequent terms (n-grams), (2) clustering of terms based of word embedding vector similarity using k-means, (3) scoring of clusters using mappings to known SMI concepts, (4) manual annotation of concepts/terms (which?) to categories. The authors then provide a detailed analysis supported by manual review by two psychiatrists. A substantial number of new relevant symptomology terms are identified through this process.\nThe authors apply standard approaches/tools for doing the text analysis, which are generally well explained and easy to follow. The authors do not directly justify the frequency floor of 10, or provide details of how many of each type of n-gram (uni/bi/tri-grams) were identified in the data. There appear to be very few trigrams, for instance. Using only frequency, how do you prevent uninteresting patterns such as \"of the\"?\nThe manual data cleaning process could have been performed semi-automatically with simple string processing tools; was this considered? (Why else would an informatician specifically need to do it?)\nThe intuitions underlying the cluster scoring functions are not clearly stated; we are told it is related to prior knowledge of concepts but it is unclear what the objectives of the specific formulas presented/used are. Why are outliers of particular interest?\nFor future work, the authors may be interested in experimenting with topic modeling rather than k-means clustering; see 1 for an approach which couples word embeddings with topic modeling. This could be more effective than k-means clustering, in particular due to the challenge of having to determine a good value for \"k\".\nThe notion of \"n-gram\" is not used entirely consistently with its broader usage in the literature; usually that refers specifically to a term of a given length (e.g. 1-grams/unigrams, 2-grams/bigrams) while different length terms are mixed here. The authors might consider using the word \"term\", or they have referred to \"concepts\" which seem to be equivalent to terms.\nRegarding the limitation that no context was provided to the annotators; would it make sense to provide a concordance of some of the instances of the terms to the annotators in future efforts?\nAlso, the IAA is tied to the 9 categories defined on page 7; where do these categories come from? Are they related to standard or validated frameworks for symptoms in psychiatric assessment? If not, why were those categories chosen?\nDid you perform any error analysis to explore the IAA further, e.g. a confusion matrix between categories? Is it possible that rather than considering these categories to be independent (the typical assumption for Cohen's Kappa) that some overlap between the categories might be expected?\nThe data is protected by patient privacy constraints and hence cannot be made openly available (indeed the authors could only work on the data in a restricted \"offline\" setting). Given the nature of the data, this is understandable. OTOH, given that the analysis largely makes use of existing code plus extensions for scoring functions, it would make sense to share the methods in an open repository.\nThe authors should include a suitable reference for PANSS. There is also a substantial literature on terminology induction (e.g. 2) which would be appropriate to reference.\nThe writing in the manuscript is generally clear, although I identified a few things that could be rephrased or clarified:\nThe word \"depiction\" seems to mean \"usage\" or \"phrase\" or \"expression\" or similar; \"depiction\" is typically used in the context of art or illustration and I found it strange in a language-expression related context. Are phenotypes and symptomatology always the same thing? Is a phenotype a set of behaviours/symptoms? The abstract is not as clear as it could be. The final sentence of the Background paragraph should use \"it is\" rather than \"it's\" but more importantly it implies that the objective is to assess clinician preferences as opposed to actual usage. Are these the same? Also, n-grams, vector space models, concepts, vocabulary and depictions are all introduced; it is a bit confusing without having read the full paper. I wonder if it could be simplified somewhat? As a nitpick, in the Introduction the authors refer to \"predicting the diversity of vocabulary\"; the work does not address prediction of vocabulary or its diversity but rather involves analysis of that vocabulary. Another nitpick in the Introduction is the use of the term \"authorship\"; I suppose the authors mean \"writing\" or \"description\" or \"summary\" or similar.\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? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3605", "date": "08 May 2018", "name": "Richard Jackson", "role": "Author Response", "response": "Thank you very much for your insightful comments. Please see our responses below: The authors apply standard approaches/tools for doing the text analysis, which are generally well explained and easy to follow. The authors do not directly justify the frequency floor of 10, or provide details of how many of each type of n-gram (uni/bi/tri-grams) were identified in the data. There appear to be very few trigrams, for instance. Using only frequency, how do you prevent uninteresting patterns such as \"of the\"? We started this project with the explicit intention of making as few assumptions as possible about semantic relationships contained within the 20 million documents in the CRIS corpus. To this end, we kept pre-processing very light, and no attempt was made to eliminate very common n-grams. Our chief assumption in planning the methodology was that uninteresting, high frequency n-grams that appear in many contexts would occupy locations in the vector space a substantial distance away from the n-grams we were interested in (symptomatology). We therefore sought to maximise the performance of our clustering method and scoring algorithm, which we hoped would filter off the n-grams that carry little information. In addition, any uninteresting patterns that did survive the filter, we removed via some simple string processing tools (see response to your additional question below on this). Regarding the counts of different n-grams, we have added the following to the vocabulary creation subsection: “ Sentences and tokens were extracted from each document using the English Punkt tokeniser from the NLTK 3.0 suite\\cite{bird_natural_2009}. Each token was converted to lower case. A vocabulary was then constructed of all 1-gram types in the corpus, supplemented with frequently occuring bi-grams and tri-grams using the Gensim\\cite{rehurek_lrec} suite and the sampling method proposed by Mikolov \\textit{et al}\\cite{mikolov2013distributed}. Bi-grams and tri-grams with a minimum frequency of 10 occurrences in the entire corpus were retained, to give a total vocabulary size of 896 195 terms (617 095 unigrams, 277 490 bigrams, 303 trigrams and 1307 non-word entities). No further assumptions about the structure of the data, such as the need for stemming/lemmatisation, were made. ” The manual data cleaning process could have been performed semi-automatically with simple string processing tools; was this considered? (Why else would an informatician specifically need to do it?) Indeed it was, although this was not made clear in the manuscript. We have added a short piece of text (in the “expert curation” subsection) to explain this: “ The contents of the top scoring clusters underwent a two stage curation process. The first stage was performed by an informatician, and involved several simple string processing tasks to filter out uninteresting n-grams. Such processes included removal of n-grams that contained tokenisation failures (for example, single character non-word tokens such as ‘y’, ‘p’) and other constructs that had low information content, such as n-grams composed of stop words. A final manual check followed to reduce the amount of annotator burden required by the clinical team. ” The intuitions underlying the cluster scoring functions are not clearly stated; we are told it is related to prior knowledge of concepts but it is unclear what the objectives of the specific formulas presented/used are. Why are outliers of particular interest? We accept this was not clearly stated and have adjusted the manuscript accordingly, in the “Vocabulary clustering and cluster scoring” section: “ With the data clustered, we sought to identify one or more clusters of interest for further examination. To this end, we devised a simple `relevance' cluster scoring approach based upon prior knowledge of common SMI symptom concepts. The intuition behind our approach is that the training of the Word2Vec model will cause n-grams that represent `known' concepts of SMI symptomatology to co-locate in close proximity to each other in vector space, and the clustering approach will place them in the same cluster, along with other n-grams that theoretically relate to these SMI symptomatology concepts. The additional contents of this cluster may therefore hold n-grams that represent concepts of SMI symptomatology undefined by our team, but in natural use by the wider clinical staff of the SLAM Trust during the course of their duties. By identifying the richest cluster(s) in terms of the known SMI symptomatology lexicon, we sought to drastically reduce the search space of n-grams in the corpus to carry forward for human assessment. ” For future work, the authors may be interested in experimenting with topic modeling rather than k-means clustering; see 1 for an approach which couples word embeddings with topic modeling. This could be more effective than k-means clustering, in particular due to the challenge of having to determine a good value for \"k\". We agree that recent advancements in topic modelling approaches are relevant to our work here. Regarding the specific case of using external word embedding models, we suspect that our target domain, (UK clinical text), is a sub-language, and the use of external word embeddings (even from very large corpora, such as Google News in the model proposed in the suggested citation) will have limited value for discovery on our data. The concepts of interest in our work are technical in nature, and seem likely to be specific to heavily regulated documents and therefore unlikely to exist in publically available datasets. On the other hand, there is no requirement to build the word embedding model from external datasets. Ultimately, there’s clearly a range of additional techniques in the literature that would be worthwhile experimenting with. We have updated our discussion as follows: “ During peer review, it was suggested that recent advancements in topic modelling approaches may be relevant to our work. Many groups have sought to combine the popular technique of Latent Dirichlet Allocation (LDA)\\cite{blei2003latent} with word embedding models to derive appropriate terminology for a given topic\\cite{cao2015novel,hinton2009replicated,srivastava2013modeling}. For instance, Nguyen et al\\cite{nguyen2015improving} propose an extension of LDA that makes use of a word embedding model trained on a very large corpus of text to improve the performance of topic coherence modelling on several datasets. Future work might seek to explore such techniques, and (assuming regulatory barriers can be overcome), the potential of creating word embedding models from very large clinical text corpora by combining data with other care organisations. “ The notion of \"n-gram\" is not used entirely consistently with its broader usage in the literature; usually that refers specifically to a term of a given length (e.g. 1-grams/unigrams, 2-grams/bigrams) while different length terms are mixed here. The authors might consider using the word \"term\", or they have referred to \"concepts\" which seem to be equivalent to terms. For technical clarity, we’ve removed references to ‘n-gram’ and replaced them with ‘term’ where appropriate. Our usage of concept refers to medical concepts (via our putative discovery process or otherwise). This is now consistent in our amendments. Regarding the limitation that no context was provided to the annotators; would it make sense to provide a concordance of some of the instances of the terms to the annotators in future efforts? We agree this would be a useful method to assist in the decision making process for manual curation, and have adjusted the text: “ The results of our IAA exercise between two experienced psychiatrists suggested a moderate level of agreement in categorising the newly identified constructs. Given that this annotation exercise did not provide any context beyond the n-gram, and that the nature of SMI symptom observation is somewhat subjective, perhaps it is to be expected that agreement was not higher. As suggested during peer review, providing a concordance of some of the instances of each n-gram, along with expert panel discussion and engagement with international collaborative efforts in SMI research may prove valuable in seeking more formal definitions of the identified constructs. ” Also, the IAA is tied to the 9 categories defined on page 7; where do these categories come from? Are they related to standard or validated frameworks for symptoms in psychiatric assessment? If not, why were those categories chosen? The categories were derived from the Shorter Oxford Textbook of Psychiatry (chapter 3, page 44), and the experience of the teams Clinical Psychiatrists. We have updated the text as follows: “ The second, more important stage was composed of independent annotation of the curated concept list by two psychiatrists, to identify likely synonyms and new symptomatology based on their clinical experience. Each concept was assigned to one of the below 8 `substantive' categories, or a 9th `other' category. The categories were derived from\\cite{harrison_shorter_2018}, and the experience of the team Clinical Psychiatrists. ” Did you perform any error analysis to explore the IAA further, e.g. a confusion matrix between categories? Is it possible that rather than considering these categories to be independent (the typical assumption for Cohen's Kappa) that some overlap between the categories might be expected? No further attempts to explore the errors in IAA were made in this analysis. Given the high level of cross-sectional and longitudinal overlap between mental disorder diagnoses classified as ‘SMI’ and the subjectivity involved in observation, it’s reasonable to think that there would be a tendency for errors to overlap in certain categories (for instance ‘insight’ and ‘cognition’). However, we think that this is outweighed by the far more complex issue of the clinical validation of the concepts we identified (which the scope of this study did not allow for). The data is protected by patient privacy constraints and hence cannot be made openly available (indeed the authors could only work on the data in a restricted \"offline\" setting). Given the nature of the data, this is understandable. OTOH, given that the analysis largely makes use of existing code plus extensions for scoring functions, it would make sense to share the methods in an open repository. We agree this would be useful, and now provide a link to a repository in the paper: “ Example code used in this analysis is available at: https://github.com/RichJackson/clustering_w2v ” The authors should include a suitable reference for PANSS. There is also a substantial literature on terminology induction (e.g. 2) which would be appropriate to reference. We think that the reviewer might have missed our original reference to the PANSS on page 3. However we mistakenly repeated the full acronym on page 10. This is now corrected. Regarding terminology induction, we have modified the following text in the introduction as follows. This now includes a reference to this article which we feel is particularly topical, given our domain: \" First, insight must be obtained regarding real-world language usage such that universally understood medical entities, encompassing hypernomy, synonymy and hyponomy adequately represent models of concepts. Similarly, because of the abundant use of acronyms in the medical domain, a large percentage have two or more meanings\\cite{liu_study_2002}, creating word sense disambiguation problems. As such, significant efforts have arisen to supplement these types of knowledge bases with appropriate real world synonym usage extracted from EHR datasets\\cite{henriksson_identifying_2013}. The problem may be considered analogous to difficulties in the recognition, classification and mapping of technical terminology variants throughout the biomedical literature, which is known to be an impediment to the construction of knowledge representation systems (see \\cite{krauthammer_term_2004} for a review). Krauthammer, Michael, and Goran Nenadic. \"Term identification in the biomedical literature.\" Journal of biomedical informatics 37.6 (2004): 512-526. \" The writing in the manuscript is generally clear, although I identified a few things that could be rephrased or clarified: The word \"depiction\" seems to mean \"usage\" or \"phrase\" or \"expression\" or similar; \"depiction\" is typically used in the context of art or illustration and I found it strange in a language-expression related context. We have rephrased this language throughout Are phenotypes and symptomatology always the same thing? Is a phenotype a set of behaviours/symptoms? Yes - the definition of a phenotype is the set of observable characteristics of an organism resulting from its genotype and interaction with its environment. We believe that symptom/behaviour profiles can be reasonably viewed in this way (and these are commonly referred to in phenotypic terms in mental health research). The abstract is not as clear as it could be. The final sentence of the Background paragraph should use \"it is\" rather than \"it's\" but more importantly it implies that the objective is to assess clinician preferences as opposed to actual usage. Are these the same? You are correct to say that ‘actual usage’ and ‘clinical preference’ are different concepts here. Our work aims to capture ‘preference’ in clinical language constructs that do not reflect matches to industry knowledge base projects (regardless of the reason). We have made several small changes in the text to reflect this. Also, n-grams, vector space models, concepts, vocabulary and depictions are all introduced; it is a bit confusing without having read the full paper. I wonder if it could be simplified somewhat? This now reads: “ By utilising a large corpus of healthcare data, we sought to make use of semantic modelling and clustering techniques to represent the relationship between the clinical vocabulary of internationally recognised SMI symptoms and the preferred language used by clinicians within a care setting. We explore how such models can be used for discovering novel vocabulary relevant to the task of phenotyping Serious Mental Illness (SMI) with only a small amount of prior knowledge. “ As a nitpick, in the Introduction the authors refer to \"predicting the diversity of vocabulary\"; the work does not address prediction of vocabulary or its diversity but rather involves analysis of that vocabulary. This sentence now reads: “ Given a sufficiently large corpus of documents, typically authored by hundreds of clinical staff over several years, it is often difficult to track the evolution of vocabulary used within the local EHR setting to describe potentially important clinical constructs. ” Another nitpick in the Introduction is the use of the term \"authorship\"; I suppose the authors mean \"writing\" or \"description\" or \"summary\" or similar. We’ve also addressed this. Thanks once again for your valuable insights" } ] } ]	1	https://f1000research.com/articles/7-210
https://f1000research.com/articles/7-540/v1	04 May 18	{ "type": "Brief Report", "title": "Long non-coding RNA LINC00987 may function as a tumor suppressor in lung adenocarcinoma", "authors": [ "Adrian Salavaty", "Zahra Rezvani", "Ali Najafi", "Adrian Salavaty" ], "abstract": "Long non-coding RNAs (lncRNAs) are a group of transcripts over 200 nucleotides in length that do not code for proteins. The association of the dysregulation of numerous lncRNAs with several malignancies, including lung cancer, has been frequently reported. This study aims to inspect the association of genomic and transcriptomic alterations to the lncRNA LINC00987 with lung adenocarcinoma, a subtype of lung cancer, using a bioinformatic approach. To this end, we used three publically available online databases, cBioPortal, the International Cancer Genome Consortium Data Portal and the GEPIA web server. In short, our results demonstrated that LINC00987 expression might have a tumor suppressive role in lung adenocarcinoma and levels of expression could be of prognostic value for this cancer type.", "keywords": [ "LINC00987", "Lung adenocarcinoma", "Tumor suppressor lncRNA", "Genotranscriptomic analysis", "Bioinformatics" ], "content": "Introduction\n\nLung cancer is the leading cause of cancer-related death in the world among both men and women1. Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer and accounts for over a million cancer-related deaths each year2,3. Thus, the identification of molecules involved in the initiation and development of LUAD should be a high priority and could help the early diagnosis, prognosis, and treatment of this lethal cancer.\n\nLong non-coding RNAs (lncRNAs) are a subclass of non-protein-coding transcripts over 200 nucleotides in length that are involved in several cancer-related biological processes, including cell growth, cell proliferation, and apoptosis4,5. It has been frequently reported that lncRNAs, due to their pivotal regulatory roles in the cellular events, especially cell cycle processes, can be used as cancer biomarkers6. HOTAIR, for example, is an oncogenic lncRNA that is being used as a biomarker for several cancers7. LINC00987 is a poorly known lncRNA that has been observed to be dysregulated in LUAD by microarray studies8. However, the genomic and transcriptomic alterations to LINC00987 lncRNA in LUAD have not yet been precisely examined.\n\nIn this study, we investigated the association of LINC00987 genomic and transcriptomic alterations with LUAD. Moreover, the relationship between the LINC00987 expression level and the overall survival (OS) of LUAD patients was inspected. To accomplish these goals, three well-known online databases, cBioPortal, the International Cancer Genome Consortium (ICGC) Data Portal and the GEPIA web server, were employed.\n\n\nMethods\n\nCopy number alterations in LINC00987 lncRNA across The Cancer Genome Atlas (TCGA) LUAD study (586 samples) were retrieved from the cBioPortal database9,10. To this end, the “Putative copy-number alterations from GISTIC” and “All Tumors (586)” were selected as the genomic profile and the patient/case set, respectively. The Gene-level Analysis tool of the TCGA Copy Number Portal (Analysis Version: 2015-06-01 stddata__2015_04_02 regular peel-off)11 was used to determine whether LINC00987 is located within or near any peak region of somatic copy-number alterations in LUAD (516 samples). TCGA Copy Number Portal facilitates the understanding of high resolution copy number data amassed from cancer samples in the TCGA. Furthermore, the mutations to LINC00987 lncRNA in LUAD was analyzed by means of the ICGC Data Portal (https://dcc.icgc.org) To this purpose, the “LINC00987” term was searched in the ICGC Data Portal and the projects related to the LUAD were selected from the results.\n\nThe GEPIA web server12 was used to investigate the differential expression of LINC00987 lncRNA between LUAD tumor (#483) and normal (#347) samples. To this end, the Differential Expression Analysis and the Expression DIY Boxplot tools were employed with default options. The association of the differential expression of the LINC00987 lncRNA with the OS of LUAD patients was examined using the Survival Plots tool of the GEPIA web server with default parameters.\n\n\nResults\n\nCopy number alteration analysis of LINC00987 demonstrated that this lncRNA was deeply deleted in over 4 percent of LUAD sequenced cases/patients (Figure 1a). However, LINC00987 lncRNA was amplified in 5 out of 230 LUAD sequenced samples. Analysis of somatic copy-number alterations to LINC00987 demonstrated that this lncRNA is located within a focal peak region of deletion (chr12:2782810-17821101) in LUAD (overall frequency of deletion: 0.2578; q-value: 0.0802). In addition, our results indicated that LINC00987 was mutated in 6.06% of LUAD donors, including chr12:g.9392556T>C, chr12:g.9392654T>C, and chr12:g.9393053C>G, according to the LUNG CANCER - KR project.\n\n(a) Copy number alterations of LINC00987 in LUAD according to the The Cancer Genome Atlas data (230 sequenced samples). Red, blue, and gray colors are indicative of amplified (n=10), deleted (n=5), and non-altered (n=215) samples, respectively. Cumulatively, 7% (n=15) of the sequenced samples had copy number alterations. The figure was retrieved from the cBioPortal database. (b) Dysregulation of LINC00987 lncRNA in LUAD. The asterisk indicates the extreme outliers. The figure was created using Boxplot function in Expression DIY of the GEPIA web server. (c) The association of LINC00987 expression level with the overall survival of LUAD patients. The figure was created using the GEPIA web server.\n\nTranscriptomic analysis of LINC00987 using the GEPIA web server showed that this lncRNA was significantly downregulated (log2(fold change) = −1.225; adjusted p-value = 1.17e−129) in LUAD (Figure 1b). Survival analysis indicated that the expression level of LINC00987 is significantly related (log-rank p-value = 0.0023) to the OS of LUAD patients (Figure 1c).\n\n\nDiscussion\n\nThe aim of this study was to examine the association of genotranscriptomic alterations of LINC00987 lncRNA with LUAD using a bioinformatic approach. There is an increasing number of publications revealing the key roles of lncRNAs in cancer development and their potential as cancer biomarker.\n\nAccording to our results, LINC00987 lncRNA is deleted in a significant proportion of the LUAD cases. LINC00987 was mutated in over 6 percent of LUAD patients. Frequent deletion and functional mutations are characteristic of dysregulated tumor suppressor genes in cancer13–15. LINC00987 lncRNA is under-expressed in LUAD samples compared with normal samples. Furthermore, survival analysis indicated that downregulation of LINC00987 lncRNA is associated with a lower OS period in LUAD patients. These cues, (that is, the under-expression of LINC00987 in cancer and the positive association of its expression level with OS of cancer patients) are characteristic of tumor suppressor genes in cancer.\n\nOverall, LINC00987 may be a tumor suppressive lncRNA in LUAD and could be considered as a biomarker for the diagnosis and/or prognosis of patients with this cancer type. However, further bioinformatic and experimental studies are required to decipher the precise function of LINC00987 in lung tissue and evaluate its application as a biomarker for LUAD.\n\n\nData availability\n\nDataset 1. The copy number alteration raw data, retrieved from the cBioPortal database. DOI: 10.5256/f1000research.14785.d20256316.\n\nAll of the results presented in this study can be directly achieved using the procedures described in the Methods section.", "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 results shown in this study are in part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/.\n\n\nReferences\n\nTorre LA, Siegel RL, Jemal A: Lung Cancer Statistics. Adv Exp Med Biol. 2016; 893: 1–19. PubMed Abstract \| Publisher Full Text\n\nChen Z, Fillmore CM, Hammerman PS, et al.: Non-small-cell lung cancers: a heterogeneous set of diseases. Nat Rev Cancer. 2014; 14(8): 535–46. PubMed Abstract \| Publisher Full Text \| Free 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\nShen XH, Qi P, Du X: Long non-coding RNAs in cancer invasion and metastasis. Mod Pathol. 2015; 28(1): 4–13. PubMed Abstract \| Publisher Full Text\n\nAnastasiadou E, Jacob LS, Slack FJ: Non-coding RNA networks in cancer. Nat Rev Cancer. 2018; 18(1): 5–18. PubMed Abstract \| Publisher Full Text\n\nBolha L, Ravnik-Glavač M, Glavač D: Long Noncoding RNAs as Biomarkers in Cancer. Dis Markers. 2017; 2017: 7243968. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHajjari M, Salavaty A: HOTAIR: an oncogenic long non-coding RNA in different cancers. Cancer Biol Med. 2015; 12(1): 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu G, Chen J, Pan Q, et al.: Long noncoding RNA expression profiles of lung adenocarcinoma ascertained by microarray analysis. PLoS One. 2014; 9(8): e104044. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGao J, Aksoy BA, Dogrusoz U, et al.: Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 2013; 6(269): pl1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCerami E, Gao J, Dogrusoz U, et al.: The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012; 2(5): 401–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeroukhim R, Mermel CH, Porter D, et al.: The landscape of somatic copy-number alteration across human cancers. Nature. 2010; 463(7283): 899–905. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTang Z, Li C, Kang B, et al.: GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 2017; 45(W1): W98–W102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalmain A, Gray J, Ponder B: The genetics and genomics of cancer. Nat Genet. 2003; 33 Suppl: 238–44. PubMed Abstract \| Publisher Full Text\n\nDong JT: Chromosomal deletions and tumor suppressor genes in prostate cancer. Cancer Metastasis Rev. 2001; 20(3–4): 173–93. PubMed Abstract \| Publisher Full Text\n\nChen J, Fu L, Zhang LY, et al.: Tumor suppressor genes on frequently deleted chromosome 3p in nasopharyngeal carcinoma. Chin J Cancer. 2012; 31(5): 215–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalavaty A, Rezvani Z, Najafi A: Dataset 1 in: Long non-coding RNA LINC00987 may function as a tumor suppressor in lung adenocarcinoma. F1000Research. 2018. Data Source" }	[ { "id": "34549", "date": "07 Jun 2018", "name": "Antonio Marco", "expertise": [ "Reviewer Expertise Non-coding RNAs", "Gene expression", "Evolution", "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\nThe authors explored major databases of genomic alterations in cancer samples and evaluated a potential role of LINC00987 in Lung Adenocarcinoma.\nThey concluded that their results \"demonstrate that LINC00987 expression might have a tumor suppressive role in lung adenocarcinoma\".\nAs an investigation evaluating the relationship between a non-coding gene an a human disease it is an interesting piece of work. However, I believe that the results are not supported and the work lacks a more appropriate statistical framework.\nThe choice of LINC00987 as a potential gene involved in this cancer is based on a paper by Xu et al. After screening the paper and the supplementary annotation I haven't found any mention to it. It may be due to a change in nomenclature, but then it must be clearly stated.\n\nThe deletion of LINC00987 is associated to a deletion of half on an arm of chromosome 12. There are hundreds of genes in the region so any association to LINC00987 must be clearly differentiated.\n\nLikewise, the expression levels can be associated to this deletion. Hundreds of other genes will be also underexpressed. This also affects the analysis of Figure 1C.\nFrom my point of view, and considering the evidence shown, there is no evidence of a causal connection between LINC00987 and Lung Adenocarcinoma. A convincing analysis should take into account the expected mutation/deletion/expression/survival profile of other genes (expectations) including those in the frequently deleted arm of chromosome 12.", "responses": [ { "c_id": "3717", "date": "08 Jun 2018", "name": "Abbas Salavaty", "role": "Author Response", "response": "We agree with you about the clarification of gene nomenclature. Actually, LINC00987 was previously known as LOC100499405. Please take a look at row 817 of the Table S5 of the paper by Xu et al. You may also get this point by checking the RefSeq transcript ID (NR_036466) of LINC00987 and LOC100499405. We will consider clarifying on this point in the next revision of the paper. According to the results obtained from the TCGA Copy Number Portal, LINC00987 is located within a focal peak region of deletion (chr12:2782810-17821101) in LUAD. Basically, this means that the gene LINC00987 and several other genes are frequently deleted in LUAD patients. However, no distinctive association between LINC00987 deletion and LUAD could be perceived from the results. It is completely true that deletion of a chromosomal region in a proportion of cells is equal to the underexpression of deleted genes. However, in this study, we specifically focused on the lncRNA LINC00987, not other genes neighboring that. Even though under-expression of other genes might affect the overall survival of LUAD patients, we specifically investigated whether the expression level of LINC00987 is associated with the overall survival of LUAD patients or not. This is a common and approved strategy and statistical analysis. Actually, this is a research note, not a comprehensive research paper, which could be an inspiring viewpoint for the conduction of further in-silico and in-vitro assays on the association of LINC00987 and LUAD. Overall, thank you for your precious time and consideration." } ] }, { "id": "36973", "date": "31 Aug 2018", "name": "Wen Cai Zhang", "expertise": [ "Reviewer Expertise Tumor initiation/progression", "cancer metabolism", "non-coding RNAs" ], "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 found that LINC00987 is a new long non-coding RNA which is poorly known in lung adenocarcinoma. Using genomic and transcriptomic databases, the authors discovered that LINC00987 is a tumor suppressive non-coding RNA and its expression levels correlate with patients’ survival. This finding is a good clue to discovering potential roles of LINC00987 in clinical diagnosis and prognosis in lung cancer patients. However, additional data are needed to claim that LINC00987 is a tumor suppressor.\nMy comments are as below.\n\nCould any known lung relevant lncRNAs (such as MALAT1) be applied and validated using the database (GEPIA)?\n\nCould the author reproduce the data shown in Fig 1b and 1c using a different database to demonstrate differential expressions of LINC00987 in lung adenocarcinoma and normal tissues and survival curve?\n\nCould the author validate the differential expression levels for LINC00987 in several lung adenocarcinoma and normal lung epithelial cell lines?\n\nFurthermore, could plasmids for LINC00987 be applied to validate the tumor suppressive roles in lung adenocarcinoma cell lines?", "responses": [] } ]	1	https://f1000research.com/articles/7-540
https://f1000research.com/articles/7-271/v1	05 Mar 18	{ "type": "Research Article", "title": "Ethanol production using vegetable peels medium and the effective role of cellulolytic bacterial (Bacillus subtilis) pre-treatment", "authors": [ "Salman Khan Promon", "Wasif Kamal", "Shafkat Shamim Rahman", "M. Mahboob Hossain", "Naiyyum Choudhury", "Salman Khan Promon", "Wasif Kamal", "M. Mahboob Hossain", "Naiyyum Choudhury" ], "abstract": "Background: The requirement of an alternative clean energy source is increasing with the elevating energy demand of modern age. Bioethanol is considered as an excellent candidate to satiate this demand. Methods: Yeast isolates were used for the production of bioethanol using cellulosic vegetable wastes as substrate. Efficient bioconversion of lignocellulosic biomass into ethanol was achieved by the action of cellulolytic bacteria (Bacillus subtilis). After proper isolation, identification and characterization of stress tolerances (thermo-, ethanol-, pH-, osmo- & sugar tolerance), optimization of physiochemical parameters for ethanol production by the yeast isolates was assessed. Very inexpensive and easily available raw materials (vegetable peels) were used as fermentation media. Fermentation was optimized with respect to temperature, reducing sugar concentration and pH. Results: It was observed that temperatures of 30°C and pH 6.0 were optimum for fermentation with a maximum yield of ethanol. The results indicated an overall increase in yields upon the pretreatment of Bacillus subtilis; maximum ethanol percentages for isolate SC1 obtained after 48-hour incubation under pretreated substrate was 14.17% in contrast to untreated media which yielded 6.21% after the same period. Isolate with the highest ethanol production capability was identified as members of the ethanol-producing Saccharomyces species after stress tolerance studies and biochemical characterization using Analytical Profile Index (API) ® 20C AUX and nitrate broth test. Introduction of Bacillus subtilis increased the alcohol production rate from the fermentation of cellulosic materials. Conclusions: The study suggested that the kitchen waste can serve as an excellent raw material in ethanol fermentation.", "keywords": [ "Bioethanol", "yeast", "cellulolytic bacteria" ], "content": "Abbreviations\n\nSC1 - yeast isolates from sugarcane juice; DJ1 - yeast isolates from date juice; pH - Negative logarithm of hydrogen ion concentration; °C - Degree Celsius; % - Percentage; CH3CH2OH - ethanol or ethyl alcohol; YEPD - Yeast Extract Peptone Dextrose; nm - Nanometer; gm - Gram; ml - Milliliter; rpm - Round per minute; v/v - volume per volume; w/v - weight per volume; spp. - Species; et al. - And others.\n\n\nIntroduction\n\nFermentation-derived ethanol (CH3CH2OH) or ethyl alcohol is commonly known as bioethanol. Ethanol can be produced chemically from petroleum, and from biomass or sugar substrates fermentation1. This organic chemical is a flammable, clear and colorless liquid which can be used as fuel. Other functions of ethanol include its use as a solvent, antifreeze and germicide2. With the aims of protecting the environment and reducing dependence on petroleum and nonrenewable energy sources, the development of renewable energy sources has become increasingly important. Several processes of bioethanol production currently exist, such as microbiological production from fermentable organic substrates or carbohydrates by yeast. Fermentation of cellulosic biomass, molasses, vegetable peels or food wastes can be considered as an economical process of bioethanol production3. Bioethanol produced from cellulosic materials by direct conversion is utilized in countries such as Brazil, Canada and, USA4. The economical production of bioethanol requires an easily available supply of inexpensive raw materials. Organic food waste is one of the topmost suitable materials for that process. Solid food wastes from household, restaurants or food processing industries can be obtained as a substrate to be used as fermentation medium for bioethanol production. Food wastes can also be recycled as animal feed and fertilizer after specific treatment.\n\nThe foremost focus of this ethanol production technology is the optimized utilization of biomass resources and microbial action on fermentation. One promising technique is the fermentation of lignocellulosic biomass where hydrolysis by specific microbial cellulase enzymes is involved5,6. Ethanol can be derived from the fermentation of sugar-containing materials. Different yeast varieties are reported for the fermentation of lignocellulosic substrates to produce ethanol7,8.\n\nThe objective of the project is to establish a highly efficient microbial fermentation process by natural yeast isolates to produce ethanol. It is to be mentioned that ethanol production rate from insoluble lignocellulosic biomass is currently not economical. Therefore, commonly available cellulosic kitchen wastes were used as raw material. Proper treatment of the substrate was done to optimize the fermentation condition which has resulted in a highly efficient and economical production rate. Potential wild-type yeast strains were isolated from date juice, sugarcane juice, grapes, and pineapples. Wild-type yeasts were identified by the biochemical and physiological characterization and taken under comparative studies and experiments to obtain a strain with high productivity. Cellulose degrading bacteria (Bacillus subtilis) was used for pre-treatment of the fermentation media. Cellulolytic microorganism debased celluloses present in the lignocellulosic fermentation media and the degraded materials were easier and more readily available to be fermented by yeast.\n\n\nMethods\n\nWild-type yeasts were isolated from sugarcane juice and date juice. Aforementioned sources were collected from the local market and kept for 1 week at room temperature for yeast growth. The samples were inoculated into YEPD (Yeast Extract Peptone Dextrose) broth which is composed of 1% yeast extract (Y1625), 2% peptone (P7750), 2% glucose or dextrose (G8270) (Sigma-Aldrich, St Louis, MO, USA) and the desired volume of distilled water. Cultures from the broth were plated on YEPD agar media and incubated at 37ºC for 48 hours. The grown colonies were cultured again on YEPD agar medium under the same growth condition to obtain isolated colonies. After the incubation, the isolated colonies (slant) were preserved at 4°C refrigeration. The culture was maintained by periodic sub-culturing.\n\nA compound microscope (Model-CX-21, Olympus, Japan) was used to observe the cell morphology and the presence of yeasts were confirmed which were isolated from sugarcane juice (Figure 1A) and date juice (Figure 1B) (named SC1 and DJ1 respectively). Identification of each isolate of yeast up to species level was carried by the methods demonstrated by Kreger-Van Rij (1984)9 based on the morphology, sporulation and fermentation characteristics, as well as the assimilation of nitrogen and a range of carbon sources. Yeast specific API® identification kit (bioMérieux, Marcy-l'Étoile, France) was also used by inoculating 48-hrs culture broths into the chambers according to the protocol.\n\nThe cell morphology (unicellular ovoid shape, multipolar budding; white and creamy texture) of yeasts under the compound microscope (100X) from sugarcane juice (A) and date juice (B).\n\nEthanol tolerance of yeast isolates was tested by inoculating isolates in YEPD broth supplemented with varying concentrations (5%, 10%, 15% and 20%) of absolute ethanol and incubated at 30°C for 48 hours. To observe the thermotolerance, the isolates were inoculated in YEPD broth and incubated at different temperatures (25°C, 30°C, 37°C and 44°C) for 48 hours. The growth of the yeast isolates at different pH was observed by inoculating isolates in YEPD broth with different pH (2–10; adjusted by adding drops of basic NaOH or acidic diluted HCl in the solution while reading a pH meter (E-201-C Shanghai Ruosuaa Technology company, China)) and incubated at 30°C for 48 hours. Initial optical densities of each tube during inoculation and optical densities after incubation were measured using the spectrophotometer (UVmini-1240 spectrophotometer, Shimadzu, Kyoto, Japan) at 600 nm against the medium as the blank.\n\nLignocellulosic biomass was used as fermentation medium. Residual waste parts of potato, papaya, pumpkin, the cucumber was used as fermentation medium. These vegetable peels were collected from households (Mohakhali area) and chopped into smaller pieces. Solid wastes (250 gm) were pulverized with 1000 ml water in an electrical blender machine. The blended material was transferred into a beaker and boiled for 10–15 minutes. Hydrochloric acid was added (2 ml) to decrease the pH to avoid bacterial contamination and convert calcium to calcium sulfate salts.\n\nYeast subcultures derived from 24–48 hours old streak plates were inoculated into 10 ml of 0.9% normal saline using a sterile loop. A cell suspension of Bacillus subtilis was prepared by inoculating 24 hours’ old culture into 10 ml NaCl (0.85%) saline. The suspensions were made homogenous using vortex machine after inoculation.\n\n150 ml fermentation media was added to 500 ml Erlenmeyer conical flasks. Cellulosic media were aseptically inoculated with Bacillus subtilis suspension and incubated for 24 hours at 37°C in shaking condition (80 rpm). After the incubation, yeast cell suspension was inoculated and the flasks were cotton plugged and incubated in a rotary incubator (WIS-20R, Wonju-si, Daihan Scientific, Korea) at 30°C for 48 hours in shaking condition (120 rpm). Yeast isolates were inoculated into another set of similar cellulosic media which were not treated with the cellulolytic organism and incubated under the aforementioned fermentation condition.\n\nInitial assay of ethanol production rates was performed by volumetric analysis in Conway units10. A fractional distillation set was used to separate ethanol from fermented broths. Samples yielding feasible results were distilled and the ethanol percentages of the distillates were determined by specific gravity using an alcohol meter (5453 Vinometer, LD Carlson, Kent, OH, United States).\n\n\nResults\n\nMorphology was visually observed as white and creamy texture, ovoid shape, multipolar budding pattern, under microscope (Figure 1A and B). Sporulation was confirmed due to the presence of ascospore. Nitrate reduction was not exhibited in the nitrate assimilation test (Figure 2A). Carbohydrate assimilation tests were conducted using API® 20C test strips (bioMérieux, Marcy-l'Étoile, France) (Figure 2B). In all cases, positive results were obtained for glucose, galactose, maltose, starch, and fructose (Table 1). Therefore, carbohydrates and nitrate assimilation test results signified the strong probability of isolates being the species Saccharomyces cerevisiae. Yeast isolates from sugarcane juice (SJ1) had a good growth at 25°C, 30°C, and 37°C, but showed poor growth at 40°C and 44°C. Yeast isolates from date juice (DJ1) had a good growth at 30°C, and 37°C, moderate growth at 40°C, but propagated poorly at 25°C and 44°C. Yeast isolate SC1 and DJ1 showed a variable growth result at pH 2–10. Overall, pH 5 and 6 was optimum growth conditions where the isolate SC1 exhibited the highest growth at pH 6 and DJ1 had its best growth at pH 5. All isolates showed excellent growth at 5% and 10% ethanol concentrations throughout the entire 48-hour incubation period (Table 2).\n\n(A) Negative nitrate reduction test indicated by the yellow color (positive test would result in red color change) (B) API 20 C X kit results for different carbohydrates fermentation using API kit after 48 hours. Color in the chamber indicates a positive result, negative results retain the yellow color of the broth. Active ingredients contained in each chamber from left to right: 0 – none, 1 – D-glucose, 2 – Glycerol, 3 – Calcium 2-keto-D-gluconate, 4 – L-arabinose, 5 – D-xylose, 6 – Adonitol, 7 – Xylitol, 8 – D-galactose, 9 – Inositol, 10 – D-sorbitol, 11 – Methyl α-D-glucopyranoside, 12 – N-acetylglucosamine, 13 – D-cellobiose, 14 – D-lactose, 15 – D-maltose, 16 – D-saccharose/Sucrose, 17 – D-trehalose, 18 – D-melezitose, 19 – D-raffinose.\n\n(Legends: + + Positive, + - Variable, -- Negative).\n\n(Legends: + + Positive, + - Variable, -- Negative).\n\nWith fermentation conditions of 30°C incubation temperature with a pH of 6, the highest rate of alcohol production from a cellulosic medium (a mixture of papaya and potato peels pretreated with Bacillus subtilis) was 14.17% v/v or 141.7 gm/L (w/v) by yeast isolate SC1 (Figure 3A; Dataset 1). On the other hand, under the same fermentation conditions, the highest rate of alcohol production using the same cellulosic medium not treated with cellulolytic bacteria was 6.21% v/v or 62.1 gm/L (w/v) by the isolate SC1 (Figure 3B; Dataset 2). The highest rate of alcohol production from the pretreated potato and papaya media was 12.24% v/v or 122.4 gm/L (w/v) by isolate DJ1 (Figure 3A; Dataset 1) under a 48-hour fermentation condition at 30°C incubation temperature. The lowest alcohol production rate recorded under the same conditions using the untreated potato and cucumber media (2.15% v/v) by the isolate DJ1 (Figure 3B; Dataset 2). Fermented media with the highest percentage of alcohol (14.17% v/v) was distilled by a fractional distillation set. This highest percentage was achieved by the yeast isolate SC1 in fermentations condition of 30°C, pH 6 at 120 rpm. The cellulosic media pretreated with Bacillus subtilis was distilled after fermentation and the distilled product (one-time distillation) had an ethanol percentage of 52% v/v. In contrast, cellulosic media which was not treated with Bacillus subtilis had an ethanol percentage of 12% v/v after the first distillation. Fermentation in other media was recorded highest at 6.23% or 62.3 gm/L (w/v) alcohol production at pH 6 by SC1 isolate after 24-hrs fermentation (Table 3).\n\nMedia treated with Bacillus subtilis (A) and No pre-treatment (B).\n\n\nDiscussion\n\nDespite the availability of several industrial strains of yeasts, local isolates are usually more adapted to their own climatic condition. In this study, yeasts were isolated from local resources. Utilization of isolated yeasts is an important strategy for the production of bioethanol11. On the basis of the white and creamy appearance of selected isolates on solid media with butyrous colony texture, polar budding and oval cellular shape it can be assumed that isolates are members of Saccharomyces spp. from the method described by Boekhout and Kurtzman (1996)12. Fermentation of different sugars by the selected yeast isolates was observed. Yeast isolates from sugarcane (SC1) utilized glucose, maltose, fructose, galactose, starch, sucrose, and arabinose but failed to grow on sorbitol, melibiose, mannitol, trehalose, inositol, xylose and lactose. Yeast isolates from date juice (DJ1) utilized glucose, maltose, fructose, galactose and starch, but failed to grow in trehalose, xylose, sucrose and lactose. The conclusion was further reinforced by biochemical tests performed using bioMérieux’ API® 20C kits. Kit results for SC1 and DJ1 indicated that all of the isolates are Saccharomyces cerevisiae. As previous studies by Ramani et al. (1998)13 indicate that API® 20C kits have a statistical accuracy of 97% for common yeasts, the conclusions were assumed to be correct. Furthermore, nitrate assimilation tests for all isolates yielded negative results which confirming our hypothesis. Thermotolerance tests also indicated that all isolates (SC1 and DJ1) grew best at 30°C within a 48-hour incubation period; this is also the optimum growth temperature of Saccharomyces cerevisiae described by Alexopoulos (1962)14. As for ethanol tolerance, the general trend observed was a decrease in terms of tolerance of all isolates above 10% ethanol concentration signified by a slowdown in growth rate with a near growth stunt at 20%. Teramoto et al. (2005)15 demonstrated that members of Saccharomyces spp. can tolerate ethanol concentrations of up to 16.5%. However, since the isolates are wild-type Saccharomyces yeasts, an average maximum tolerance of 10% ethanol does not mean that they cannot be members of Saccharomyces spp. Different growth factors affect the pH tolerance of yeast. It was reported by Ivorra et al. (1999)16 that the optimum pH range for ideal growth varies from 4–6 depending on the strain. The cellular structure of yeast has a diverse mechanism to endure pH. In this experiment, yeast isolates SC1 and DJ1 had a variable growth result from pH 2–10. Both of the isolates had excellent growth from pH 4 to 6. However, those isolates were able to grow at all the pH condition, but pH lower than 3 and higher than 7 was not suitable for a good growth. Overall, pH 5 and 6 were optimum growth conditions where isolate SC1 had its best growth at pH 6 and isolate DJ1 had its best growth at pH 5. The ethanol production rate was recorded from the fermentation of different cellulosic media after 24 and 48-hours fermentation. The production rate ranged from 2.15% or 21.5 gm/L to 14.17% v/v or 141.7 gm/L (w/v). Isolate SC1 had the highest rate of ethanol production (14.17% v/v), and isolate DJ1 had the lowest rate of ethanol production (2.15% v/v) in shaking condition at 30°C with a media pH of 6. Ethanol production rate was also observed in shaking condition at 30°C with a media pH of 5. In this condition, isolate SC1 had the highest rate of ethanol production (9.42% v/v) and isolate DJ1 had the lowest rate of ethanol production (2.17% v/v), which surpassed the previous reports17–19. Ethanol production using kitchen waste media has exceeded the earlier works20. In a study, Nofemele et al. (2012)21 demonstrated 7.8% percent ethanol production from sugarcane molasses using Saccharomyces cerevisiae. In Bangladesh, five yeast isolates were reported22 to be used for the similar experiments where those isolates (TY, BY, GY-1, RY and SY) had alcohol production rate of 12.0%, 5.90%, 5.80%, 6.70% and 5.80%, respectively at 30°C after 48 hours of incubation. Significant elevation of ethanol production rate was observed in the co-fermentation process where cellulosic media were inoculated with cellulolytic bacteria previously. Overall, the method proves the efficiency of the co-fermentation23–25.\n\n\nConclusions\n\nThe present study allowed the isolation and characterization of two Saccharomyces cerevisiae isolates (SC1 and DJ1) with potential for ethanol production. Yeast isolated from sugarcane juice (named SC1 for this study) showed the highest percentage of alcohol production from cellulosic substrates. Vegetable peels pretreated with cellulolytic bacteria are detected as a suitable fermentation substrate. If the fermentation conditions are optimized, this procedure may be used for large-scale bioethanol production from cellulosic wastes. Scaling up of the experiment can be beneficial for power generation, bioethanol can be used as an alternative to fossil fuels. The raw materials required for the production of bioethanol are cheap and available. It will decrease environmental pollution, pave the pathway towards a proper waste management system and also fertilizers can be produced from the wasted substrate.\n\n\nData availability\n\nDataset 1: Alcohol production from vegetable peels by yeast isolates SC1 and DJ1 at pH 6. Media pre-treated with Bacillus subtilis. 10.5256/f1000research.13952.d19563926\n\nDataset 2: Alcohol production from vegetable peels by yeast isolates SC1 and DJ1 at pH 6. Media without Bacillus subtilis pre-treatment. 10.5256/f1000research.13952.d19564027", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was conducted under a grant (39.009.002.01.000.053.2014-2015/16/BS-31) of Ministry of Science and Technology (Government of Bangladesh).\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\nAuthors are grateful to (Late) Prof. Dr. A. A. Ziauddin Ahmad, Chairperson, Department of MNS, BRAC University, Dhaka, for allowing the research work. Their humble regards go to Akhtaruzzaman Khan and Abira Khan and also thankful to Md. Rafid Feisal, Sreoshee Rafiq and Tribeni Ghosh for their help and support.\n\n\nAuthor information\n\nWhen the research was carried out, SSR was a MSc student at Biotechnology Program, Department of Mathematics and Natural Sciences, BRAC University, and NC was the Coordinator of Biotechnology and Microbiology programmes at Department of Mathematics and Natural Sciences, BRAC University.\n\n\nReferences\n\nHossain N, Zaini JH, Mahlia TM: A Review of Bioethanol Production from Plant-based Waste Biomass by Yeast Fermentation. Int J Technol. 2017; 8(1): 5–18. Publisher Full Text\n\nLicht FO: World Ethanol Market: The Outlook to 2015. Agra Europe Special Report, Tunbridge Wells, UK, 2006; Accessed 12 Sep 2017. Reference Source\n\nPeris D, Moriarty RV, Alexander WG, et al.: Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production. Biotechnol Biofuels. 2017; 10(1): 78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThenmozhi R, Victoria J: Optimization and improvement of ethanol production by the incorporation of organic wastes. Pelagia Research Library. 2013; 4(5): 119–23. Reference Source\n\nYamada R, Nakashima K, Asai-Nakashima N, et al.: Direct ethanol production from ionic liquid-pretreated lignocellulosic biomass by cellulase-displaying yeasts. Appl Biochem Biotechnol. 2017; 182(1): 229–37. PubMed Abstract \| Publisher Full Text\n\nLee YG, Jin YS, Cha YL, et al.: Bioethanol production from cellulosic hydrolysates by engineered industrial Saccharomyces cerevisiae. Bioresour Technol. 2017; 228: 355–61. PubMed Abstract \| Publisher Full Text\n\nChandra RP, Bura R, Mabee WE, et al.: Substrate pretreatment: the key to effective enzymatic hydrolysis of lignocellulosics? Adv Biochem Eng Biotechnol. 2007; 108: 67–93. PubMed Abstract \| Publisher Full Text\n\nWang J, Hu M, Zhang H, et al.: Converting Chemical Oxygen Demand (COD) of Cellulosic Ethanol Fermentation Wastewater into Microbial Lipid by Oleaginous Yeast Trichosporon cutaneum. Appl Biochem Biotechnol. 2017; 182(3): 1121–30. PubMed Abstract \| Publisher Full Text\n\nKreger-Van Rij NJ: The Yeast a Taxonomic Study. New York: Elsevier Science Publishing Company. 1984; 1082. Reference Source\n\nConway RK, McClelland J, Shapouri H: Comments Concerning the Environmental Protection Agency’s Regulation of Fuels and Fuel Additives: Renewable Oxygenate Requirement for Reformulated Gasoline Proposed Rule. Public Document A-93-49. U.S. Department of Agriculture, Office of Energy; 1994; Accessed 15 Sep 2017.\n\nJayakody LN, Ferdouse J, Hayashi N, et al.: Identification and detoxification of glycolaldehyde, an unattended bioethanol fermentation inhibitor. Crit Rev Biotechnol. 2017; 37(2): 177–89. PubMed Abstract \| Publisher Full Text\n\nBoekhout T, Kurtzman CP: Principles and methods used in yeast classification, and an overview of currently accepted yeast genera. In: Wolf K, editor. Nonconventional Yeasts in Biotechnology: A Handbook. Springer-Verlag, Berlin, Heidelberg; 1996; 1–99. Publisher Full Text\n\nRamani R, Gromadzki S, Pincus DH, et al.: Efficacy of API 20C and ID 32C systems for identification of common and rare clinical yeast isolates. J Clin Microbiol. 1998; 36(11): 3396–98. PubMed Abstract \| Free Full Text\n\nAlexopoulos CJ: Sub-class hemiascomycetidae, the yeast and leaf-curl fungi. In: Introductory Mycology. Second Edition. Toppan Printing Company, Japan; 1966; 241–58.\n\nTeramoto Y, Sato R, Ueda S: Characteristics of fermentation yeast isolated from traditional Ethiopian honey wine, ogol. Afr J Biotechnol. 2005; 4(2): 160–3. Reference Source\n\nIvorra C, Pérez-Ortín JE, del Olmo M: An inverse correlation between stress resistance and stuck fermentations in wine yeasts. A molecular study. Biotechnol Bioeng. 1999; 64(6): 698–708. PubMed Abstract \| Publisher Full Text\n\nRahman SS, Hossain MM, Choudhury N: Effect of Various Parameters on the Growth and Ethanol Production by Yeasts Isolated from Natural Sources. Bangladesh J Microbiol. 2013; 30(1–2): 49–54. Publisher Full Text\n\nNasir A, Rahman SS, Hossain MM, et al.: Isolation of saccharomyces cerevisiae from pineapple and orange and study of metal’s effectiveness on ethanol production. Eur J Microbiol Immunol (Bp). 2017; 7(1): 76–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahman SS, Sarkar MK, Islam MR, et al.: Isolation of yeasts from raisins and palm-juice and ethanol production in molasses medium. Indian J Sci Technol. 2016; 9(12). Publisher Full Text\n\nRahman SS, Hossain MM, Choudhury N: Bioethanol fermentation from kitchen waste. Under Review. 2018.\n\nNofemele Z, Shukla P, Trussler A, et al.: Improvement of ethanol production from sugarcane molasses through enhanced nutrient supplementation using Saccharomyces cerevisiae. J Brewing Distilling. 2012; 3(2): 29–35. Reference Source\n\nKhan AR, Malek MA, Choudhury N, et al.: Alcohol production from molasses and liquid sugar using some indigenous yeast isolates. Bangl J Microbiol. 1989; 6(1): 37–42.\n\nLee CR, Sung BH, Lim KM, et al.: Co-fermentation using Recombinant Saccharomyces cerevisiae Yeast Strains Hyper-secreting Different Cellulases for the Production of Cellulosic Bioethanol. Sci Rep. 2017; 7(1): 4428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi YJ, Lu YY, Zhang ZJ, et al.: Co-fermentation of Cellulose and Sucrose/Xylose by Engineered Yeasts for Bioethanol Production. Energy Fuels. 2017; 31(4): 4061–7. Publisher Full Text\n\nCasa-Villegas M, Marín-Navarro J, Polaina J: Synergies in coupled hydrolysis and fermentation of cellulose using a Trichoderma reesei enzyme preparation and a recombinant Saccharomyces cerevisiae strain. World J Microbiol Biotechnol. 2017; 33(7): 140. PubMed Abstract \| Publisher Full Text\n\nPromon SK, Kamal W, Rahman SS, et al.: Dataset 1 in: Ethanol production using vegetable peels medium and the effective role of cellulolytic bacterial (Bacillus subtilis) pre-treatment. F1000Research. 2018. Data Source\n\nPromon SK, Kamal W, Rahman SS, et al.: Dataset 2 in: Ethanol production using vegetable peels medium and the effective role of cellulolytic bacterial (Bacillus subtilis) pre-treatment. F1000Research. 2018. Data Source" }	[ { "id": "31498", "date": "16 Mar 2018", "name": "Nazia Hossain", "expertise": [ "Reviewer Expertise Biofuel and bioenergy" ], "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\nAbstract\n- Kitchen waste might be a possible raw material for bioethanol production for clean technology according to the research result but it cannot be concluded as excellent source. Authors need to upgrade this view for the abstract (conclusion section).\n\nIntroduction\n- Please provide a brief description regarding the necessity of biofuel and bioethanol application in the beginning. - You may describe the common reactions what may take place for ethanol production from biomass and the role of microbes. - I suggest splitting first paragraph into 2 paragraphs and describing specific issues for each instead of mixing up.\n\nMethods\n- Ok.\n\nResult and Discussion\n- Did you isolate yeast from local resources due to economic purpose? If so, please mention it. - Sometimes fermentation might be effective after 48 hours (e.g. 72hr or 96hr). Please explain why this study have not tried upto 96 hr or above. Do your have any recommendation to do so in future? - Usually supplement addition (e.g. MgSO4) enhances fermentation process and increases overall bioethanol production. May you please elaborate why this study has not applied any nutritional component? - In discussion section, authors cited ‘Teramoto et al. (2005) demonstrated that members of Saccharomyces spp. can tolerate ethanol concentrations of up to 16.5%’ which is from 2005. Many studies have been performed with Saccharomyces spp. after 2005 what proved more than 50% bioethanol production. For instance, I suggest authors to check (Sugar and Bioethanol Production from Oil Palm Trunk (OPT) by Nazia Hossain & Rafidah Jalil). - Please provide few examples of comparative studies and compare your results with others since vegetable and fruits peels are very common sources of bioethanol generation worldwide.\n\nConclusion\n- The study result showed little amount of bioethanol production rate what is not favourable for economical view but environmental. So authors simply cannot assume that this procedure may be used for large-scale application without any specific modification or upgrade. In that case, author might recommend some factors to accelerate the production rate before scale-up. - I answered ‘Partly’ to the question ‘Are the conclusions drawn adequately supported by the results?’ because limitations are not mentioned in the conclusions.\n\nOverall\n- Bioethanol is more appropriate than ethanol (In Title). - Grammar needs to be re-checked. - Many poorly structured sentences were being visible in the whole manuscript. Therefore, overall english should be improved. - I answered ‘Partly’ to the question ‘Is the work clearly and accurately presented and does it cite the current literature?’ as authors cited lot of old references while many upgraded experiments have been performed later on. I suggest authors to use references after 2000 only (especially for discussion part).\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? Partly", "responses": [ { "c_id": "3630", "date": "03 May 2018", "name": "Shafkat Shamim Rahman", "role": "Author Response", "response": "Thanks to Dr. Hossain for her constructive comments. We conducted a number of changes in response to her suggestions. Abstract (conclusion section) is corrected. The first paragraph of the introduction is modified by splitting and reorganizing. The reason to isolate yeast from local resources is added in the discussion. Our previous studies (Rahman et al. 2013; Nasir et al. 2017) in the same lab showed the alcohol production reached the peak at 48 hours. After 96 hours, the rate was either same or dwindled (possibly ethanol inhibition). So this study was designed for 48 hours. Addition of supplements usually increases the fermentation rate (Nasir et al. 2017), which was overlooked in the study design. We recommend adding this in future works. We express gratitude to enlighten us on the higher ethanol tolerance, which we believe will be helpful in future optimization studies. After 2000 references and comparison studies are added in the discussion. Limitations and recommendations are also added in the conclusion. The title of the article has also amended." } ] }, { "id": "32524", "date": "20 Apr 2018", "name": "Md Fakruddin", "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\nReference should be added in method sections.\n\nEthanol production was represented in both % and gm/L. Please use only one unit uniformly.\n\nLong time fermentation (96 hr) should be done to ensure the efficiency of the isolate.\n\nIntroduction should be improved to address the objective of the study.\n\nCompare your results with related published papers.\n\nPlease indicate the advantage of kitchen waste over more economical source such as molasses.\n\nIt is not clear what changes occur due to bacillus pretreatment.\n\nWithout large scale fermentation, the conclusion may be misleading.\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?\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": [ { "c_id": "3629", "date": "03 May 2018", "name": "Shafkat Shamim Rahman", "role": "Author Response", "response": "We thank Dr. Fakruddin for his critical evaluation to improve the article. This report helps us to add few points to increase the quality of the article. We added new references in the methods section. In the preliminary submission, the results presented in percentage. In the revised article, gm/L (specific gravity reading) was also added to meet the editorial review requirement. Our previous studies (Rahman et al. 2013; Nasir et al. 2017) in the same lab showed the alcohol production reached the peak at 48 hours. After 96 hours, the rate either remain same or dwindled (possibly ethanol inhibition). So this study was designed for 48 hours. We added another objective in the introduction. We compared the results of tolerance tests with Teramoto et al. 2005, Ivorra et al. 1999 and results of fermentation to Nofemele et al. 2012, Khan et al. 1989, Rahman et al. 2013 and Nasir et al. 2017 in the discussion section. Now, we also added a comparison with related published paper. The advantage of kitchen waste over molasses is added at the end of the discussion. Figure 3 described the after effect of pretreatment, which resulted in increased ethanol production. Conclusions are ameliorated. If there is any specific recommendation, it would be helpful to conduct the revision." } ] } ]	1	https://f1000research.com/articles/7-271
https://f1000research.com/articles/7-538/v1	03 May 18	{ "type": "Research Article", "title": "Validity and reliability of the Thai version of the Autism Treatment Evaluation Checklist: A two-phase diagnostic accuracy study", "authors": [ "Kanitha Sunakarach", "Pattapong Kessomboon", "Kanitha Sunakarach" ], "abstract": "Background: This study aimed to evaluate the psychometric properties of the Thai version of the Autism Treatment Evaluation Checklist (Thai-ATEC); a tool which has been developed for Thai parents and caregivers who have children with autism spectrum disorder (ASD). Methods: Approval for this study was first obtained from the appropriate Ethics committee and from the original Autism Treatment Evaluation Checklist (ATEC) developers. This was a two-phase study. Phase 1 consisted of the forward–backward translation of the ATEC and phase 2 included the testing of psychometric properties, i.e. the validity and reliability of the final draft of the tool. The validity of the tool was assessed by comparing Thai-ATEC scores of parents and caregivers of 160 children with ASD with the assessment of a child and adolescent psychiatrist using DSM-V criteria on the same group of children. The inter-rater reliability of the tool was tested using a two-way model of intra-class correlation coefficient (ICC) for two-parent/caregivers’ assessment of 50 children with ASD. Results: The validity of the Thai-ATEC was moderate to high. A cut-off point of ≤38 scores was used to distinguish between children with ASD with mild symptoms and the rest of the children (sensitivity = 94%, specificity= 61.9%, and the area under ROC curve = 90%). A cut-off point of ≥68 scores was used to distinguish between children with ASD with a severe degree of symptoms and the rest (sensitivity = 94%, specificity = 62.8%, area under receiver operating characteristic curve = 85%). The inter-rater reliability was very strong (ICC = 0.97). Conclusions: The Thai-ATEC has moderate to high validity and high reliability.", "keywords": [ "Autism Treatment Evaluation Checklist", "Thai version Autism Treatment Evaluation Checklist", "validity", "reliability", "autism spectrum disorder" ], "content": "Introduction\n\nAutism spectrum disorders (ASD) are a cluster of neurodevelopmental disorders that are characterized by abnormalities in social interactions and verbal and nonverbal communication, and restricted, repetitive and stereotyped patterns of behavior, interests and activities1. ASD symptoms begin in the early years after birth, generally before 3 years of age2. The prevalence of ASD has increased over recent years in many parts of the world3. In Thailand, it was estimated that there were 180,000 children with ASD nationwide (i.e. 2.8 per 1,000 population or 15 per 1,000 children age <15)4. However, this may underestimate the true scale. Accurate diagnosis needs adequately trained health professionals and appropriate tools5.\n\nMany assessment tools have been developed to screen and diagnose children with autism, such as the Autism Diagnostic Interview-Revised (ADI-R)6, the Pre-Linguistic Autism Diagnostic Observation Schedule (PL-ADOS)7, and the Childhood Autism Rating Scale (CARS)8. These tools are mostly used by intensively trained mental health professionals. These tools are time-consuming, however; for example, the ADI-R takes 2 h to complete and the PL-ADOS takes 30 min. Scores derived from the tools had low sensitivity with older children with ASD9 and these tools are not readily available to caregivers. Although these tools are appropriate for research, because of these limitations, other tools have been investigated that have been standardized for evaluating the changes of symptoms of children with autism, one example of which is the ATEC10.\n\nThe ATEC was developed by Rimland and Edelson at the Autism Research Institute11. The tool has been designed to enable parents to assess their children with ASD. It has increased the effectiveness of home caring because it allows for confirmation of the effectiveness of treatments provided10,12. This tool can be used by parents, caregivers, teachers, professionals and researchers. The ATEC has been translated to 18 languages and is widely used globally10,13.\n\nThe ATEC is a questionnaire consisting of four subscales: 1) speech/language/communication (14 items); 2) sociability (20 items); 3) sensory/cognitive awareness (18 items); and 4) health/physical/behaviour (25 items). The ATEC total score ranges from 0 to 179 with maximum scores on the subscales of 28 (speech/language/communication), 40 (sociability), 36 (sensory/cognitive awareness), and 75 (health/physical/behavior). The higher the subscale and total scores, the more impaired the child.\n\nAfter testing 1,358 children, results showed a high internal consistency (Pearson correlation coefficient, split-half method) of the total ATEC scores (r=0.94) and high internal consistencies of the subscales (speech/language: r=0.92; sociability: r=0.84; sensory/cognitive awareness: r=0.88; health/physical behavior: r=0.82)14. Compared with the CARS utilized by a health care professional, the result showed that ATEC scores and CARS scores had a significant correlation (ρ =0.71, p-value <0.0001)15. It also showed that the predictive value of the ATEC was good. This could be used to continuously follow up the response to treatments10.\n\nIn Thailand, there is a high need for an efficient assessment tool, and a high interest in the ATEC. However, no formal standardized translation of the original ATEC has been done and no research to assess its psychometric properties in the Thai context has been conducted. Therefore, this study aimed to translate the ATEC into Thai and evaluate its psychometric properties.\n\n\nMethods\n\nThis was a two-phase study. Phase 1 consisted of the forward–backward translation of the ATEC and phase 2 included the testing of psychometric properties, i.e. the validity and reliability of the final draft of the tool. The full study protocol can be accessed at the Khon Kaen University graduate school or from the corresponding author upon request.\n\nThe authors obtained permission from Dr. Stephen M. Edelson, the original developer of the ATEC at the Autism Research Institute (San Diego, CA, USA), for the use of the ATEC in this study. The forward–backward translation included six processes: 1) studying and gaining understanding of the assessment tool, after which a child psychiatrist work at Child and Adolescent Mental Health Rajanagarindra Institute, Bangkok and clinical psychologist work at Psychiatric Hospital Nakhon Ratchasima Rajanagarindra translated it into the Thai language; 2) synthesis of the first translation; 3) backward translation into the English language by a British translator who had been living in Thailand for over 10 years(Private business at his home in Bangkok), and a professional translator Director and Tutor at Samarthome, Nakhonratchasima, both employed for this particular task; 4) comparing the back translation with the original version for content, sematic, idiomatic, and cultural equivalences by an expert committee comprising six members, including the child psychiatrist, the clinical psychologist, two backward translators, an expert in communication with children with ASD work at Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, and a parent of a child with ASD with a high English proficiency, Vice president Autism Society of Thailand, Bangkok; 5) trying out the assessment tool, during which the processes were undertaken iteratively until the tool had reached acceptable understandability by stakeholders; and 6) submitting the final tool and documentations to the original ATEC developers. The complete Thai-ATEC can be found in Supplementary File 1.\n\nThe validity of the tool was assessed by comparing the Thai-ATEC scores from parents and caregivers of 160 children with ASD, with the assessment of the same child and adolescent psychiatrist using DSM-V criteria2 of the same group of children with ASD. The psychiatrist was blinded to the results of the parallel measure.\n\nThe validity indexes included sensitivity, specificity, predictive value, likelihood ratio positive (LR+), likelihood ratio negative (LR−), accuracy, and area under receiver operating characteristic (ROC) curve. These were calculated by exploring an appropriate cut-off point score that was able to distinguish between children with ASD with mild degree of symptoms and the rest of the children. Another appropriate cut-off point score was assessed for its ability to distinguish children with ASD with a severe degree of symptoms from the rest of the children.\n\nThe tool’s inter-rater reliability was tested by comparing the Thai-ATEC scores from 50 pairs of parents/caregivers of 50 children with ASD. Each pair of parents/caregivers was independently assess the child with ASD.\n\nInclusion criteria were parents/caregivers of children with ASD who were able to read and write. The age of the children with ASD was ≥3 years old. The sample size was calculated based upon data from a pilot study with expected sensitivity = 0.81, expected specificity = 0.60, prevalence = 0.4, desired pecision = 0.10 and a confidence level = 95%. Therefore, 160 parents and caregivers of children with ASD were needed. They were recruited simple random sampling during an appointment with the psychiatrist at the outpatient clinic at the North Eastern Institute of Child and Adolescent Mental Health, Khon Kaen province, Thailand, during May–August 2017.\n\nThe tool’s inter-rater reliability was tested using a two-way model of intra-class correlation coefficient (ICC) rater assessment of the same children with ASD. The participants of the inter-rater reliability test consisted of 50 pairs of parents and caregivers who were subsets of the 160. Each pair was selected for this test because both were caring for the same child and accompanied the child on the day of the assessment. Therefore, a child with one parent or one caregiver on those days at the hospital was not selected. After a child was examined by the psychiatrist, each pair of parents and caregivers was invited into a separate room and informed about how to complete the Thai-ATEC. The pairs were not allowed to meet and talk to each other. There was no time limit to fill in the checklist. Parents and caregivers spent an average of 9.21 minutes completing the tool.\n\nThe validity indexes including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, likelihood ratio (LR+/−), area under receiver operating characteristic (ROC) curve and the appropriate cut-off point of the tool were calculated by comparing scores obtained from the parents’ and caregivers’ Thai-ATEC assessment with the results of the psychiatrist’s assessment using DSM-V criteria. The 95% confidence intervals of these indexes were also estimated. An appropriate cut-off point score was explored based on its ability to distinguish between children with ASD with mild degree of symptoms and the rest of the children. Another appropriate cut-off point score was assessed for its ability to distinguish children with ASD with a severe degree of symptoms from the rest of the children. The tool’s inter-rater reliability was calculated using a two-way model of intra-class correlation coefficient (ICC) rater assessment of the same children with ASD. The SPSS statistical software version 19.0 was used to perform these data analyses.\n\nApproval for the study was obtained from the Khon Kaen University Ethics Committee in Human Research (HE601001). Parents of children with ASD provided written informed consent for their participation in the research.\n\n\nResults\n\nA total of 160 parents/caregivers met the criteria of selection. Most parents/caregivers were female (113, 70.6%). Their ages ranged from 18–60 years old. Most were mothers (82, 51.2%). Most of the ASD children were male (102, 63.7%). The average age of children with autism was 7.86 years old (SD = 3.43, min = 3 years, max = 18 years).\n\nAt a cut-off point of a score of ≤38, to distinguish between children with ASD with mild symptoms and the rest of the children with moderate to severe symptoms, it was found that the tool’s sensitivity was 94% (95% CI, 0.91–0.98), the specificity was 61.9% (95% CI, 0.48–0.78), the PPV was 88.3% (95% CI, 0.82–0.93), the NPV was 81.2% (95% CI, 0.67–0.94), the accuracy was 86.9%, the LR+ was 2.59, the LR− was 0.07, and the area under the ROC curve was 90% (Table 1).\n\nPPV, positive predictive value; NPV, negative predictive value; LR+, likelihood ratio positive.\n\nAt a cut-off score of ≥68 to distinguish between children with ASD with a severe degree of symptoms and the rest of the children with mild to moderate symptoms, it was found that the tool’s sensitivity was 91.3% (95% CI, 0.67–0.98), the specificity was 62.8% (95% CI, 0.55–0.74), the PPV was 28.8% (95% CI, 0.17–0.39), the NPV was 95.7% (95% CI, 0.91–0.99), the accuracy was 68.1%, the LR+ was 2.40, the LR− was 0.26, and the area under ROC curve was 85% (Table 2).\n\nPPV, positive predictive value; NPV, negative predictive value; LR+, likelihood ratio positive.\n\nTherefore, the above findings suggest that children with ASD can be assessed using the Thai-ATEC and the severity of ASD symptoms can be grouped according to the cut-off points found in this study as follows:\n\nLevel of mild symptoms: Thai-ATEC scores in the range of 1–38\n\nLevel of moderate symptoms: Thai-ATEC scores in the range of 39–68\n\nLevel of severe symptoms: Thai-ATEC scores in the range of 69–179\n\nThe flow of participants can be seen in Supplementary File 2.\n\nICCs were calculated to evaluate the inter-rater reliability of the tool. It was found that ICC for the total score was 0.97 (95% CI, 0.97–0.98). When we analyzed each subset of the tool, that is speech/language/communication, sociability, sensory/cognitive awareness and health/physical/behavior, it was found that the ICCs were 0.94, 0.86, 0.89 and 0.97, respectively. These were all more than 0.75, meaning that the assessment was highly consistent16 (Table 3).\n\nThe first observer was one of the parent/caregiver pair, and the second observer was the other person in the parent/caregiver pair.\n\nICC, intra-class correlation coefficient.\n\n\nDiscussion\n\nTo our knowledge, this was the first study of its kind that attempted to formally translate the ATEC into Thai. Standardized and rigorous translation processes were employed. Beaton et al. proposed guidelines for the process of cross-cultural adaptation of self-report measures, including forward and backward translation to ensure that tools are appropriate and comparable18. This study has intentionally followed this guideline and employed additional processes which were needed. A child and adolescent psychiatrist, a Thai professional translator, a professional ASD communicator, health professionals responsible for the care of children with ASD, a British professional translator and a parent with a high English proficiency who had a child with ASD were all involved in the translation process to ensure that the final tool was of high quality and that the meaning of the Thai-ATEC was consistent with the original ATEC tool.\n\nThe psychometric properties of the Thai-ATEC were moderate to high. The sensitivity of the tool was 94%, its specificity was 62%, and the area under ROC curve was ≥85%. The discovered cut-off points were used to divide the severity of ASD symptoms into three levels: mild, moderate and severe. These levels mild were similar to the original ATEC, However, the results showed that moderate and severe levels were different from the original14. (Table 4).\n\nA comparison of mild, moderate and severe symptoms between the original and new Thai version of the ATEC.\n\nParents and caregivers can use the ATEC to monitor children with ASD to assess the severity of impairment in different domains and the progress in response to intervention over time. The ATEC covers not only behavioral issues, but also health and systemic issues, such as sleep problems, seizures, eating, gastrointestinal problems, hyperactivity, self-injuries and sleep disturbance. Health and systemic issues are of high concern among parents19.\n\nA study in India comparing the CARS and clinical diagnoses found that at a CARS score of ≥33, the sensitivity of the scale was 81.4%, the specificity was 78.6% and the area under the curve was 81%16. Thus, the Thai-ATEC had higher sensitivity but lower specificity than the Indian CARS. Geier et al. found that the CARS correlated well with the ATEC15.\n\nThe inter-rater reliability of the Thai-ATEC was very strong. The ICC of the total score was 0.97 and the ICCs of subscale 1, 2, 3 and 4 were 0.94, 0.86, 0.84 and 0.97, respectively. The Indian study that compared the CARS with clinical diagnoses by psychiatrists16 found that inter-rater reliability was only 0.74. An ICC of >0.9 is perceived as indicating high reliability20.\n\nLimitations of this study included the time consuming nature of the processes, owing to the limited number of child and adolescent psychiatrists involved in the study. Child and adolescent psychiatrists are rare in Thailand.\n\nIn this study, the experienced child and adolescent psychiatrist who acted as the gold standard, followed the DSM-V criteria and spent 5–10 minutes per person. This relatively short length of time might have caused some mistakes and may have led to misclassification of symptoms that could have affected sensitivity and specificity. However, the single-psychiatrist approach taken in this study may have minimized subjective differences in scoring methods and provided superior consistency to a multi-psychiatrist approach.\n\nThis study was conducted in the north-eastern part of Thailand. Therefore, further studies are needed to explore the usability of the new Thai-ATEC in other parts of Thailand, where local dialects are present.\n\n\nConclusion\n\nThe Thai-ATEC has moderate to high validity and high reliability. The cut-off points at scores of ≤38 and ≥68 were appropriate to distinguish the level of severity between the mild, moderate, and severe ASD symptoms. Parents and caregivers of children with ASD can efficiently use the tool to assess their children at home and communicate the results with health professionals.\n\n\nData availability\n\nDataset 1. The Thai-ATEC scores for each child from each parent/caregiver and the corresponding diagnosis from the child psychiatrist. DOI: 10.5256/f1000research.14537.d20217517.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe study received partial financial support from the Graduate School of Khon Kaen University.\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 wish to thank all the parents, caregivers and children who participated in the Thai-ATEC study. We would also like to thank all the translators and stakeholders who were involved in the study. We sincerely thank Dr. Charnnarong Chaiudomsom and Dr. Suthra Auapisithwong for their hard work in making this study possible. We very much appreciate the strong support provided by the director and staff of the North Eastern Institute of Child and Adolescent Mental Health in Khon Kaen. We express our gratitude to S. M. Edelson of the Autism Research Institute for the permission to use the original ATEC and invaluable suggestions he gave for this study.\n\n\nSupplementary material\n\nSupplementary File 1. The Thai-Autism Treatment Evaluation Checklist questionnaire used in the current study.\n\nClick here to access the data.\n\nSupplementary File 2. Flow diagram of participants.\n\nClick here to access the data.\n\n\nReferences\n\nAmerican Psychiatric Association: Diagnostic and statistical manual of mental disorder: DSM-IV-TR. 4th ed. Washington, DC: APA; 2000. Reference Source\n\nAmerican Psychiatric Association: Diagnostic Criteria From DSM-5. Washington, DC: APA; 2013. Reference Source\n\nWorld Health Organization: Meeting report: autism spectrum disorders and other developmental disorders: from raising awareness to building capacity. 2013 Sep 16–18; Geneva, Switzerland. [cited 2017 Jan 22]. Reference Source\n\nKopetz PB, Endowed ED: Autism worldwide: prevalence, perceptions, acceptance, action. J Soc Sci. 2012; 8(2): 196–201. [cited 2017 Dec 15]. Publisher Full Text\n\nNational Institute for Health and Clinical Excellence: Autism: Recognition, Referral and Diagnosis of Children and Young People on the Autism Spectrum. London: NICE; 2011. PubMed Abstract\n\nLord C, Rutter M, Le Couteur A: Autism Diagnostic Interview-Revised: a revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders. J Autism Dev Disord. 1994; 24(5): 659–85. PubMed Abstract \| Publisher Full Text\n\nLord C, Risi S, Lambrecht L, et al.: The Autism Diagnostic Observation Schedule-Generic: a standard measure of social and communication deficits associated with the spectrum of autism. J Autism Dev Disord. 2000; 30(3): 205–23. PubMed Abstract \| Publisher Full Text\n\nSchopler E, Reichler RJ, DeVellis RF, et al.: Toward objective classification of childhood autism: Childhood Autism Rating Scale (CARS). J Autism Dev Disord. 1980; 10(1): 91–103. PubMed Abstract \| Publisher Full Text\n\nGreen J, Charman T, McConachie H, et al.: Parent-mediated communication-focused treatment in children with autism (PACT): a randomised controlled trial. Lancet. 2010; 375(9732): 2152–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMagiati I, Moss J, Yates R, et al.: Is the Autism Treatment Evaluation Checklist a useful tool for monitoring progress in children with autism spectrum disorders? J Intellect Disabil Res. 2011; 55(3): 302–12. PubMed Abstract \| Publisher Full Text\n\nRimland B, Edelson SM: Autism Treatment Evaluation Checklist (ATEC) [online]. 2000; [cited 2016 Dec 20]. Reference Source\n\nWeiner RH, Greene RL: Intention-based therapy for autism spectrum disorder: promising results of a wait-list control study in children. Explore (NY). 2014; 10(1): 13–23. PubMed Abstract \| Publisher Full Text\n\nJarusiewicz B: Efficacy of neurofeedback for children in the autistic spectrum: a pilot study. J Neurother. 2002; 6(4): 39–49. Publisher Full Text\n\nAutism Research Institute: Studies confirm validity of ATEC - 2018. 2018; [cited 2018 Jan 10]. Reference Source\n\nGeier DA, Kern JK, Geier MR: A Comparison of the Autism Treatment Evaluation Checklist (ATEC) and the Childhood Autism Rating Scale (CARS) for the Quantitative Evaluation of Autism. J Ment Health Res Intellect Disabil. 2013; 6(4): 255–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRussell PS, Daniel A, Russell S, et al.: Diagnostic accuracy, reliability and validity of Childhood Autism Rating Scale in India. World J Pediatr. 2010; 6(2): 141–7. PubMed Abstract \| Publisher Full Text\n\nSunakarach K, Kessomboon P: Dataset 1 in: Validity and reliability of the Thai version of the Autism Treatment Evaluation Checklist: A two-phase diagnostic accuracy study. F1000Research. 2018. Data Source\n\nBeaton DE, Bombardier C, Guillemin F, et al.: Guidelines for the process of cross-cultural adaptation of self-report measures. Spine (Phila Pa 1976). 2000; 25(24): 3186–91. PubMed Abstract \| Publisher Full Text\n\nDale E, Jahoda A, Knott F: Mothers' attributions following their child's diagnosis of autistic spectrum disorder: exploring links with maternal levels of stress, depression and expectations about their child's future. Autism. 2006; 10(5): 463–79. PubMed Abstract \| Publisher Full Text\n\nNunnally JC: Psychometric theory. 2nd ed. New York: McGraw-Hill; 1978." }	[ { "id": "33726", "date": "29 May 2018", "name": "Mark R Geier", "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 researchers have undertaken an important and timely study to evaluate the validity and reliability of the Autism Treatment Evaluation Checklist (ATEC) for diagnosis and evaluate children diagnosed with autism in Thailand. They have demonstrated that they were able to translate the ATEC from English into the Thai language, and then utilize the ATEC as a means to identify and evaluate the severity of children diagnosed with autism in Thailand. They have utilized appropriate methods for data collection and data analyses. The study results are consistent with previously cited published studies examining the validity of the ATEC on different populations using different metrics. The researchers appropriately consider study limitations, and the study conclusions are supported by the data.\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": "34245", "date": "11 Jun 2018", "name": "Manote Lotrakul", "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 advantages of the ATEC are that it is shorter than other measures, easy to understand and designed to be completed by parents, teachers, or caregivers, making it a suitable tool for a country where there is a shortage of psychiatrists. It provides quantitative measurements on important domains of ASD severity which increased the effectiveness of home-base care for children with ASD.\nThe authors have translated the ATEC into Thai following Beaton's Guidelines for the cross-cultural translation and adaptation of self-report instruments. The psychometric properties of the Thai-ATEC have been tested and reported as acceptable to good. The methods section and results of the study have sufficient and clear details. The authors are skeptical whether the Thai-ATEC can be used in other parts of Thailand but this may not be the case because the wording in each item is natural, clear and easy to understand.\n\nComments:\n\nPlease change “DSM-V” to “DSM-5“\n\nIt would be better if the author also present the other type of reliability, i.e., the internal consistencies of the ATEC scores and subscales.\n\nPage 6 paragraph 4: It is not relevant to compare the Thai-ATEC with the CARS study in India as the purpose of that study was to develop the screening tool whereas the ATEC is designed to measure ASD severity.\n\nPage 6 paragraph 6: This research is time consuming or slow-paced but it is not the limitation of the study.\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": "3754", "date": "21 Jun 2018", "name": "Pattapong Kessomboon", "role": "Author Response", "response": "Dear Reviewer (Manote Lotrakul),Thank you very much for the review. We would like to respond to the comments as follows:1. Yes. Please change \"DSM-V\" to \"DSM-5\"2. The internal consistencies as measured by Cronbach's alpha coefficient of the Thai-ATEC total score and the sub-scales are as follows: Total score (alpha=0.946); speech/language (alpha=0.930); sociability (alpha=0.845); sensory/cognitive awareness (alpha=0.884) and health/physical/behavior (alpha=0.842). 3.The authors agree with the reviewer on this point.4.The authors agree with the reviewer on this point.Yours sincerely,Pattapong KessomboonKanitha Sunakarach" } ] } ]	1	https://f1000research.com/articles/7-538
https://f1000research.com/articles/7-537/v1	03 May 18	{ "type": "Opinion Article", "title": "Cell line access to revolutionize the biosimilars market", "authors": [ "Dzintars Gotham" ], "abstract": "Biologic drugs are notoriously expensive. Biosimilars, though priced lower, are also costly. Analysis of the cost of production of biologics suggests that the cost of manufacture is in many cases less than 10% of the price in high-income countries, and less than a third of the price of biosimilars in India. This in turn implies that the relatively high prices of biosimilars are largely due to the need to undertake laborious reverse-engineering and phase 3 trials to demonstrate clinical similarity. In this article, it is proposed that originators could be required to submit cell line stocks to regulators and disclose details of manufacturing processes. These would be shared with prospective non-originator manufacturers to greatly reduce the investments needed to bring a non-originator biologic to market. This system would allow far greater price reductions for biologics after the expiry of monopoly rights (e.g. patents), while maintaining the monopoly rights used to incentivize drug development.", "keywords": [ "biologics", "biosimilars", "economics", "pharmacology", "access to medicines", "regulatory policy", "pharmaceutical policy", "health policy" ], "content": "Introduction\n\nThe importance of biologics in both clinical and economic terms continues to grow1. At the same time, biologics are notoriously expensive, and challenges in affordability remain even after the expiry of monopoly rights. Price reductions seen with biosimilars are in general smaller than for small molecule medicines and have a higher starting point, with originator prices typically being in the tens of thousands of dollars. Even after originator patent expiry, biosimilars strain health budgets and remain inaccessible for many patients globally2,3.\n\n\nBiologic monopoly rights and biosimilar development\n\nBiologic drugs are large, complex molecules whose exact composition is often not fully known, and the exact manufacturing process used is closely related to safety and efficacy – including the specific growth media, post-translational modifications, and the purification processes used. Details on these manufacturing processes are trade secrets and are thus inaccessible to competitors for a potentially indefinite period. Moreover, the cell line that produces the originator product remains the property of the originator like any other physical asset, and almost all information about the cell line remains a trade secret. Consequently, biosimilar manufacturers have to undertake laborious reverse-engineering work and phase 3 trials to demonstrate that their product is not different to the originator’s in a clinically significant way.\n\nThis represents a significant duplication of efforts and means that the barriers to competition in biologics far exceed the time-limited monopoly rights – granted through patents, market exclusivity, and data exclusivity – that attempt to legislatively balance the need to incentivize drug development against the social benefit of competition-driven price reductions. These barriers translate to higher costs: While the exact numbers are not publicly available, the cost of bringing a biosimilar to market in the US has been estimated to be $100–200 million, compared to $1–5 million for small-molecule medicines4.\n\nAside from development expenditures, what are the inherent costs of manufacturing a biosimilar once it has been approved? For monoclonal antibodies – the most common type of biologic drug – they are reported to range US$20,000–300,000 per kilogram of active ingredient, which includes capital investments in manufacturing facilities and overheads5,6. Multiplying these estimates by per-treatment dosage, the costs of manufacturing alone appear to represent only a small proportion of the price – 0.001–6% of current lowest prices in the US and 0.004–14% of prices in the UK, for blockbuster biologics (Table 1). Even in India, where the costs of bringing a biosimilar to market are considered to be significantly lower, and all the prices shown are for biosimilars, the estimated cost of manufacturing would represent only 0.02–27% of the price.\n\nThe table illustrates the large differences between current prices and the cost of manufacturing the active ingredient, for blockbuster biologic medicines.\n\n*Biosimilar.\n\nDetails on indications, dosage, and price sources are available in Supplementary File 1.\n\nPrices for the UK may not include confidential rebates.\n\nIt thus appears that if the need for investments in reverse-engineering and phase 3 trials were removed, price reductions for biologic therapies following the expiry of monopoly rights could be far greater than at present.\n\n\nA proposal: cell line access\n\nIn order to lower the barriers to bringing follow-on biologic products to market, prospective manufacturers should be given access to the originator cell lines and detailed descriptions of manufacturing processes.\n\nTo achieve this, originator biologics manufacturers could be required to submit a living vial of the cell line used to manufacture the product upon regulatory approval, to be stored by the relevant regulatory body. Cloned cell lines would then be shared with prospective manufacturers, prior to the expiry of the originator’s monopoly rights. Detailed descriptions of originators’ manufacturing processes, which are already submitted to regulators confidentially, could be made publicly available7.\n\nThis system would allow originators to fully enjoy legislatively granted time-limited monopoly rights, while avoiding arguably unnecessary and duplicative drug development and enabling maximal competition as soon as monopoly rights expire.\n\nI refer to this proposed system as ‘cell line access’ and biologics developed through this system as CLA biologics. The non-profit group Knowledge Ecology International has previously made a similar proposal8, and Price and Rai have proposed that incentive mechanisms could be set up to encourage the disclosure of trade secrets pertaining to manufacturing processes7.\n\n\nThe benefits of cell line access\n\nFirst, access to cell lines and production processes would dramatically reduce the financial and time investments required for developing a non-originator biologic product. The prices of CLA biologics would be expected to be substantially lower than the price reductions currently seen for biosimilars, both due to increased competition and lower costs.\n\nSecond, there would be clinical benefits, as CLA biologics could be expected to automatically be interchangeable with the originator product, rather than only ‘similar’.\n\nThird, biopharmaceutical science more broadly would benefit, especially if access to cell lines and manufacturing processes were extended also to research institutions.\n\n\nAdapting existing regulatory procedures\n\nThe potential benefits of cell line access would in large part depend on regulators being satisfied that if an identical cell line and manufacturing process are used, phase 3 trials would not be necessary, and approval could be granted based on demonstration of equivalence by other methods.\n\nDifferences between CLA biologics and the reference (originator) product would be expected to be of the same magnitude as batch-to-batch variation in the originator’s product. As originators regularly make slight changes to their manufacturing process, regulatory procedures are already in place to ensure that these changes do not jeopardize efficacy or safety9, for which in vitro analytical tools are nearly always sufficient9,10. These established procedures could be adapted to the task of ensuring that CLA biologics are not substantially different from the originator product.\n\nSimilarly, the change of manufacturer, while preserving the cell line and manufacturing processes used, is comparable to situations where originator companies use contract manufacturing organizations or regional manufacturing partners, an increasingly common practice11.\n\n\nConclusion\n\nEnabling prospective non-originator manufacturers to access originator cell lines and manufacturing processes could remove the need for laborious reverse-engineering and duplicative clinical trials. This would allow full enjoyment by originators of the monopoly rights granted through patents and other protections, while allowing far greater price reductions upon expiry of monopoly rights. While significant political challenges would be expected, this proposed mechanism could be explored further in terms of the potential economic and health impacts, and practical options for implementation.\n\n\nData availability\n\nNo data is associated with this article.", "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 author thanks Melissa J Barber for their thoughtful discussions of the ideas proposed in the article.\n\n\nSupplementary material\n\nSupplementary File 1. This document describes the data sources for the US, UK, and India prices given in Table 1, as well as assumptions regarding dosage.\n\nClick here to access the data.\n\n\nReferences\n\nBelloni A, Morgan D, Paris V: Pharmaceutical Expenditure And Policies. OECD Health Working Papers, 2016. Publisher Full Text\n\nBlackstone EA, Fuhr JP: The economics of biosimilars. Am Health Drug Benefits. 2013; 6(8): 469–478. PubMed Abstract \| Free Full Text\n\nQuintilesIMS: The Impact of Biosimilar Competition in Europe. 2017; Accessed April 12, 2018. Reference Source\n\nFederal Trade Commission Report: Emerging Health Care Issues: Follow-on Biologic Drug Competition. 2009; Accessed April 12, 2018. Reference Source\n\nKelley B: Industrialization of mAb production technology: the bioprocessing industry at a crossroads. mAbs. 2009; 1(5): 443–452. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu C, Morrow J, eds: Biosimilars of Monoclonal Antibodies: A Practical Guide to Manufacturing, Preclinical, and Clinical Development. Hoboken, New Jersey: John Wiley & Sons, Inc; 2017. Publisher Full Text\n\nPrice WN 2nd, Rai AK: Drug Development. Are trade secrets delaying biosimilars? Science. 2015; 348(6231): 188–189. PubMed Abstract \| Publisher Full Text\n\nSinghroy D: Policies about access to knowledge, data and materials to make it easier to make biosimilar drugs. May 2, 2017. Presented at the WHO Consultation on Biosimilar Drugs.\n\nMcCamish M, Woollett G: Worldwide experience with biosimilar development. mAbs. 2011; 3(2): 209–217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nU.S. Food and Drug Administration: Guidance for Industry: Q5E Comparability of Biotechnological/Biological Products Subject to Changes in Their Manufacturing Process. Accessed March 16, 2018. Reference Source\n\nThe Hindu Business Line: Emcure to make, market Roche products. Accessed March 25, 2018. Reference Source" }	[ { "id": "34142", "date": "31 May 2018", "name": "Amit Misra", "expertise": [ "Reviewer Expertise Pharmaceutics", "drug and antigen delivery systems", "innate immunity", "tuberculosis", "access to medicines", "drug regulation", "pharmaceutical policy" ], "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\nMy compliments to the author for this potentially revolutionary idea. However, solely addressing the drug regulator and enjoining it to implement cell line access (CLA) is, in my view, a sub-optimal strategy. First, the regulator may very often cite lack of capacity to preserve and distribute vials containing live cells. Issues related to refusal of access (e.g., on grounds of lack of competence or non-compliance with Good Manufacturing Practices) to applicants would require the regulator to expend a great deal of time and effort in deciding who gets a vial and who doesn't. Last, the innovator is denied any say in the dissemination of the innovation for public good if the drug regulator is the sole arbiter of CLA. With competitive marketing practices being what they are, it is not inconceivable that a competitor may choose to acquire CLA, only to generate a sub-optimal product that could then be faulted for poor performance to build a negative perception about the innovator. In my view, it would be important to invoke a \"multi-stakeholder initiative\" (MSI) involving drug regulators, civil society organizations working on access to medicines, and corporations that discover and produce biologics to ensure CLA worldwide. \"Corporate Social Responsibility\" (CSR) guidance and legislation in various countries would provide incentive to corporations to join such an MSI. A transparent process whereby applicants requesting access could be assessed and granted (or refused) CLA would be, in my view, a more sustainable strategy. Originators could then also \"hand-hold\" successful applicants through the process of adapting the cell lines to their specific bioreactors and downstream processing equipment. Of course, drug regulators would be required to notify CLA as mandatory after the monopoly period is over, while granting marketing permission (as also retrospectively for off-patent biologics). I would like to request the author to consider the feasibility and sustainability of the CLA initiative before concluding that it is sufficient for the drug regulator to require originators to share cell lines and manufacturing processes as a \"practical option for implementation.\" Finally, although it is beyond the scope of the present article, I would like to suggest that the author may touch upon the regulatory requirement of \"sameness\" in respect of biologics, and whether Phase 3 \"comparative\" clinical trials actually make sense. There is extensive literature on these aspects, including two articles cited below.\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? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "34194", "date": "12 Jul 2018", "name": "François Bocquet", "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 opinion paper is interesting. My first concern is the presentation of the production costs of the active substances.\nIndeed, only reasoning (ie for each molecule) in proportion to the Kelley figure of $20,000 to $300,000 per kilogram of active ingredient (equivalent to $ 0.02-0.3 per milligram) without having any idea of the real cost for each molecule can be misleading, and is not very satisfactory. These data being confidential, thank you to the authors to specify this in the legend of the table and not only in the supplementary material.\nEven if the idea proposed by the authors is interesting intellectually, it clashes with the respect of the patent right. It should be emphasized more in the paper.\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/7-537
https://f1000research.com/articles/7-302/v1	09 Mar 18	{ "type": "Research Article", "title": "Virus-like particles and enterovirus antigen found in the brainstem neurons of Parkinson’s disease", "authors": [ "Robert R. Dourmashkin", "Sherman A. McCall", "Neil Dourmashkin", "Matthew J. Hannah", "Sherman A. McCall", "Neil Dourmashkin", "Matthew J. Hannah" ], "abstract": "Background: In a previous study on encephalitis lethargica, we identified an enterovirus in autopsy brain material. Transmission electron microscopy (TEM), immunohistochemistry (IHC) and molecular analysis were employed. Our present objective was to investigate, using a similar approach, as to whether virus-like particles (VLP) and enterovirus antigen are present in Parkinson’s disease (PD) brainstem neurons. Methods: Fixed tissue from autopsy specimens of late onset PD and control brainstem tissue were received for study. The brain tissue was processed for TEM and IHC according to previous published methods. Results: We observed VLP in the brainstem neurons of all the cases of PD that were examined. In the neurons’ cytoplasm there were many virus factories consisting of VLP and endoplasmic reticulum membranes. In some neurons, the virus factories contained incomplete VLP. Complete VLP in some neurons’ virus factories had an average diameter of 31 nm, larger than control brain ribosomes. In the nuclei, there were VLP with an average diameter of 40 nm. In cases of human poliomyelitis, there were cytoplasmic virus factories and intranuclear virus particles similar to those observed in PD. On preparing PD brain sections for IHC there was positive staining using anti-poliovirus antibody and anti-coxsackie antibody. This result was statistically significant. Conclusions: We present evidence for an enterovirus infection in PD. For future studies, virus isolation and molecular analysis is suggested.", "keywords": [ "Virus-like particles", "disease", "electron microscopy", "immunohistochemistry" ], "content": "Introduction\n\nFollowing our study on encephalitis lethargica1, we were struck by the similarilities of the clinical course of encephalitis lethargica and Parkinson’s disease (PD), leading us to study the latter disease. We have applied the methodology used to characterise encephalitis lethargica by means of TEM of fixed brain tissues and IHC. Our aim is to identify and characterise virus particles and viral products in PD brain.\n\nPD is widespread, especially among the elderly. It was called `the shaking palsy’ by Parkinson in 18172, and it remains a common cause of debilitation and death. Two hundred years later, we present evidence suggesting an enterovirus infection is present in the brainstem in PD.\n\nFollowing the pandemic of encephalitis lethargica during 1918–1925, many of the survivors of the acute phase of the disease developed a syndrome similar to PD, but in which the patients became somnolent. They developed tremor and oculogyric crises during which their eyes rolled uncontrollably3. This syndrome was termed `post-encephalitic parkinsonism’ and more popularly in England `the sleepy sickness’. It was very common and often conflated with PD. By the end of the 20th century every patient who suffered from post-encephalitic parkinsonism had died, whereas PD continued to occur.\n\nA number of previous studies have proposed a virus etiology for PD. A review by Jang et al.4 discussed the neurological sequelae of infection by influenza virus, as well as that of other viruses known to induce parkinsonism, including Coxsackie, Japanese encephalitis B, St. Louis, West Nile and HIV viruses. Jang et al. in an in vivo study5 showed that a pathogenic influenza virus can enter the central nervous system causing neuroinflammation and induce neurodegeneration. Zhou et al.6 in a literature review suggested that viruses may be associated with neurodegenerative diseases, including Alzheimer’s disease, PD and multiple sclerosis. Duvoisin7 reported in a review the evidence for association of PD with infectious diseases. Hawkes et al.8 proposed a dual hit theory for the pathogenesis of infection in PD, in which channels for virus infection pass through the nasal mucosa and the intestinal epithelium. Svensson et al.9 and Liu et al.10 found a reduced incidence of PD in patients who underwent total vagotomy, suggesting a pathway for infection via the vagus nerve. Gibbs and Gajdasek11 carried out experiments designed to isolate a virus in PD using in vitro cell cultures co-cultivated with PD brain tissue. Direct inoculation of PD brain tissue to non-human primates was also carried out. No evidence of PD developed in the inoculated animals and no evidence was found of virus replication in the cell cultures. Wetmur et al.12 did not detect nucleic acids complementary to herpes simplex type 1 DNA or influenza A/NWS RNA in the brain of patients with sporadic PD. Elizan et al.13 reported that tests were negative for arbovirus antibody using sera from cases of encephalitis lethargica and PD, and in another study14 Elizan et al. searched for antibodies to herpes simplex virus 1, measles and rubella in the serum and cerebral spinal fluid of post-encephalitic parkinsonism patients with negative results. Schwartz and Elizan15 did not detect virus particles by means of transmission electron microscopy (TEM) in PD brain tissue nor in cell cultures co-cultivated with PD brain tissue. The possible involvement of enterovirus in PD was not considered in these studies11–15.\n\nImmature dopamine neurons grafted to PD patients showed that Lewy bodies developed in the grafts16. Jucker and Walker17 found that misfolded α-synuclein can develop disease propagating properties. Human gut microbiota from PD patients induced enhanced motor dysfunction in mice18, suggesting that gut microbiota play a part in the pathogenesis of PD.\n\nDavie, in a review of PD19, reported that the LRRK 2 gene (PARK 8) is the most common genetic cause of familial (early onset) PD. The frequency of LRRK 2 mutations in patients with a family history of PD is 5–7%. The heterozygous mutation, 2877510 G>A is the most commonly described, accounting for the majority of familial cases and up to 1.6% of cases of sporadic PD. Mok et al.20 found that, in a combined analysis of genome-wide association data of 9,387 cases of PD, eight cases were reported with deletions at the 22q1.2 chromosome site. Age of onset of PD of the patients carrying these deletions was lower (median age, 37 years) than those who did not carry the deletions (median age, 61 years). In the present study, all the PD cases are from the late age of onset.\n\nPathological studies on the incidence of Lewy bodies in the brain were carried out by Herva and Spillantini21 and Barker and Williams-Grey22, who showed that Lewy bodies were found in 6–8% of autopsies of patients with no clinical evidence of brain disease. The finding of Lewy bodies is the histopathological hallmark of PD; they carry an accumulation of α-synuclein. Barker and Williams-Grey22 reviewed the literature defining α-synuclein as the core protein of Lewy bodies. They are present in PD, in dementia with Lewy bodies, and in multiple system atrophy. Josephs and Parisi23 found that brain tissue from post-encephalitic parkinsonism cases did not carry α-synuclein protein, thus differentiating encephalitis lethargica and the subsequent syndrome, post-encephalitic parkinsonism, from PD.\n\nDrug induced parkinsonism was described as an acute/subacute syndrome in which tremor is infrequent compared to PD. Most cases of drug induced parkinsonism were caused by both classic and second generation neuroleptics and calcium channel blockers. Resolution by 75% of drug induced parkinsonism occurred six months after withdrawal of the drug24. Thalidomide may worsen existing PD25.\n\n\nMethods\n\nBrain tissue from 14 sporadic cases of PD and seven control cases was examined by TEM in order to identify virus-like particles (VLP) in the neurons of the PD brainstem. None of the control cases had poliomyelitis. The method used for TEM of autopsy brain that had been fixed and embedded in paraffin blocks for histology has been previously reported1. Fifty nm sections were cut on a Reichart ultramicrotome, stained with 1% uranyl acetate, then stained with Reynold’s lead citrate, and examined in a JEM1400 TEM (JEOL UK) at Public Health England, Colindale, UK. Images were acquired with an AMT XR60 digital camera (Deben Ltd, UK). VLP in the digital images of cytoplasmic virus factories of PD cases and ribosomes in the cytoplasm of control cases were measured using the measurement facility of the AMT image acquisition software (Deben Ltd. UK). Calliper measurements were also made of each of 30 particles in magnified paper prints of each image (Table 1). Biased selection of particles for measurement was avoided by measuring all the clearly imaged particles in each print.\n\n30 particles were measured for each case. The identification of the images is indicated by their author’s TEM serial number. The images used for measurement in Table 1 are illustrated in Figure 3, Figure 4, Figure 14 and Figure 15.\n\nClinical reports of the PD cases received from the Armed Forces Institute of Pathology (USA) and the John Radcliffe Hospital (UK) stated that Lewy bodies were found by the examining neuropathologists in the brainstem neurons of all the PD cases studied, indicating that PD was present at the time of death.\n\nIHC testing was carried out by exactly the same technique described previously1. Rabbit polyclonal anti-polio antiserum raised against polio types 1, 2, and 3, and goat anti-coxsackie polyclonal antiserum raised against a coxsackie virus isolate were donated by the Department of Virology, National Institute for Biological Standards and Control, Potters Bar, UK.\n\nMethod for preparing the polyclonal antibodies.\n\nThe inoculation of one rabbit with Poliovirus type 1 for the production of polyclonal antibody:\n\nVirus/adjuvant injected subcutaneously into loose skin, 0.25ml into 4 sites [total 1ml]. Poliovirus Sabin type 1 (1010 pfu/ml) 1:5 dilution\n\n1 rabbit for each virus receives:\n\nInoculation 1:\n\n0.5ml virus preparation + 0.5ml TiterMax Gold adjuvant, injected subcutaneously into loose skin, 0.25ml into 4 sites [total 1ml].\n\nInoculation 2:\n\n3 weeks after 1st injection: 0.5ml virus preparation + 0.5ml incomplete TiterMax Gold adjuvant, injected subcutaneously into loose skin, 0.25ml into 4 sites [total 1ml].\n\nInoculation 3:\n\n3 weeks after 2nd injection: 0.5ml virus preparation + 0.5ml incomplete TiterMax Gold adjuvant, injected subcutaneously into loose skin, 0.25ml into 4 sites [total 1ml].\n\nSample bleed: after 2 weeks\n\nFollowed by terminal bleed 13 days later.\n\nTiterMax© Gold Adjuvant Sigma Aldrich #T2684\n\nAll Plastic Luer lock syringes #Z248010\n\n3-way luer lock stopcock #S7521\n\nThe polyclonal antisera were absorbed 5x with normal human autopsy brain tissue. Histologic sections of 12 controls and 21 PD cases were stained with rabbit anti-polio polyclonal antibody, and 14 controls and 19 PD cases were stained with goat polyclonal anti-coxsackie antibody. A series of two-fold dilutions of each antibody was used. The sections were then exposed to biotinylated secondary antibody, then treated with Vector preformed avidin:biotin:enzyme complex26. The slides were then rinsed and stained with Vector Nova Red26, cleared and mounted.\n\nFurther IHC tests on PD sections and control sections of normal brain tissue were carried out using the following antibodies: mouse monoclonal antibodies to Enterovirus type 71, Enterovirus VP1 convalescent serum, mouse monoclonal antibody to human parvovirus B19, and serum from human parvovirus infection.\n\nThe statistics were calculated using Microsoft Excel 10. The Fisher test results were checked using the ‘R’ statistical package, version 3.3.3. The chi-squared tests were checked against the calculator on http://turner.faculty.swau.edu/mathematics/math241/materials/contablecalc/.\n\n\nResults\n\nTEM changes were observed in the brainstem neurons in the PD cases and in the spinal cord motor neurons in the poliomyelitis cases. TEM of PD neurons at low magnification showed advanced apoptosis. There were almost ‘empty’ nuclei with clumped chromatin and multiple cytoplasmic virus factories. Few cytoplasmic organelles remained (Figure 1 and Figure 2). VLP were found by TEM in the nuclei and cytoplasm in the neurons of all the PD cases studied. The VLP were similar in morphology to the VLP we described in the brain of encephalitis lethargica, which had been confirmed to be a strain of enterovirus by molecular analysis1. The cytoplasmic virus factories in PD neurons consisted of large numbers of VLP interspersed with irregularly shaped endoplasmic reticulum membranes and embedded in virus factory (Figure 3 and Figure 4). VLP were observed attached to the membranes (Figure 3). The average measurements of the cytoplasmic VLP in Figure 3 and Figure 4 were both 31 nm (Table 1). Cytoplasmic virus factories in other PD neurons consisted of incomplete VLP at an early stage of assembly (Figure 5, see Discussion).\n\nLow magnification of a neuron in the substantia nigra. The nucleus is almost devoid of chromatin and the remaining chromatin is clumped. Many virus factories are present in the cytoplasm (arrows). The black granules in the cytoplasm contain melanin. A high magnification of this image that shows intranuclear VLP is in Figure 4.\n\nLow magnification of a neuron in substantia nigra. Arrows point to virus factories.\n\nA virus factory in PD. There are endoplasmic reticulum membranes in this image (arrows). There are virus-like particles (VLP) that are larger than the ribosomes in control Nissl bodies. The mean diameter of the VLP in this image was 31 nm (see Table 1). The magnification has been adjusted to 200,000x so as to make the comparison of the diameters of VLP and that of the ribosomes more apparent. The magnification bar for 100 nm for these images = 20 mm.\n\nIn the cytoplasm of a PD case, the virus factories consisted of masses of virus-like particles (VLP) in an amorphous matrix. Mean size of VLP: 30 nm (see Table 1). The magnification has been adjusted to 200,000x so as to make the comparison of the diameters of VLP and that of the ribosomes more apparent. The magnification bar for 100 nm for these images = 20 mm.\n\nHigh magnification of a virus factory in a PD neuron showing VLP embedded in virus factory and membrane proliferation.\n\nIntranuclear VLP were found in all 14 cases of PD. The VLP were lined along the internal membrane of neurons and to a lesser degree throughout the nuclei (Figure 6 and Figure 7). The intranuclear VLP had an average diameter of 40 nm. The shape of the VLP was symmetrical and oval (the latter, the effect of thin sectioning) with clear edges.\n\nThere are intranuclear VLP lining the internal face of the nuclear membrane of the nucleus. The nuclear membrane is indicated by a thick arrow. VLP are demonstrated by thin arrows.\n\nVLP in a PD neuron, close to the internal face of the nuclear membrane (arrow).\n\nIn the cases of human poliomyelitis, motor neurons of the spinal cord showed cytoplasmic virus factories and apoptosis similar to that shown in PD neurons (Figure 8–Figure 10). Intranuclear virus particles were found that were similar to the VLP in morphology and diameter to those found in the brain of the PD cases (Figure 11).\n\nLow magnification of a motor neuron. Cytoplasmic virus factories are shown (arrows). The nucleus shows severe apoptosis.\n\nThis image shows cytoplasmic virus factories (arrows).\n\nA cytoplasmic virus factory, showing strands of endoplasmic reticulum and incomplete virus particles embedded in amorphous matrix.\n\nIntranuclear virus particles are shown (arrow).\n\nA Lewy body is illustrated from a case of PD (Figure 12). In this image, the nucleus and cytoplasm of the neuron was completely destroyed. There were no VLP associated with the Lewy bodies.\n\nThe cytoplasmic constituents of the cell have degenerated. No VLP were found associated with the Lewy bodies.\n\nIn the control cases, the neurons showed little apoptosis. Nissl bodies were present consisting of rough endoplasmic reticulum (Figure 13). Free ribosomes were also found (Figure 14 and Figure 15). Intranuclear VLP were found in one control case. The neurons in this case showed a moderate degree of apoptosis and lacked cytoplasmic virus factories. The finding of VLP in a control case in this study is in accordance with the observations quoted above, 21,22, in that Lewy bodies were found in the brain of a proportion of control cases. The case reported here could represent a preclinical PD event.\n\nControl image is presented of cytoplasm of brainstem neuron in a case of myocardial infarction and perforated gastric ulcer. Part of a cytoplasmic Nissl body consisting of endoplasmic reticulum is shown. The nuclear membrane is indicated (arrow).\n\nCytoplasmic ribosomes in a control neuron at high magnification. The mean diameter of the ribosomes is 20 nm (see Table 1). The magnification has been adjusted to 200,000x so as to make the comparison of the diameters of VLP and that of the ribosomes more apparent. The magnification bar for 100 nm for these images = 20 mm.\n\nImage of control brainstem neuron showing cytoplasmic ribosomes (see Table 1). The magnification has been adjusted to 200,000x so as to make the comparison of the diameters of VLP and that of the ribosomes more apparent. The magnification bar for 100 nm for these images = 20 mm.\n\nIHC staining of PD brain sections showed 13 positive results and 8 negative results using rabbit anti-polio antiserum and 13 positive and 7 negative results using goat anti-coxsackie antiserum; and for control brain sections, there were 10 negative results and 2 positive results for poliovirus antiserum and there were 13 negative results and 1 positive result for coxsackie antiserum. The data for the IHC tests are reported in Table 2 and Table 3. The results were statistically positive for the IHC staining of PD brain tissue as compared with that of control tissue (see Statistical analysis section).\n\nSample methodology for production of anti-polio virus antiserum (NIBSC).\n\nThe results were negative for IHC staining of PD tissue using mouse monoclonal anti-enterovirus antibodies as well as monoclonal and polyclonal anti-parvovirus antibody staining. The absence of reaction of enterovirus monoclonal antibodies to PD tissue sections may be attributed to the fact that the solitary epitope to which the antibodies were raised was inappropriate for the staining of PD neurons carrying the putative enterovirus antigen.\n\nA light microscopy image of an area of the inferior olive of a case of PD is shown that was stained for poliovirus antigen (Figure 16). There is heavy staining of the neurons and light staining of the surrounding neuropil.\n\nIllustrated here is an immunohistochemical image of the inferior olive of a neuron using rabbit anti-polio antibody (see Methods), showing heavy staining of neurons and lighter staining of surrounding neuropil.\n\nThe first null hypothesis, based on the data in Table 1, was that there is no difference between the measured mean size of complete cytoplasmic VLP in each of the PD cases and of cytoplasmic ribosomes in the controls. A t-test for two independent sample means without assuming equality of variances was used. The resulting probabilities are shown in Table 4. The results are all significant to at least p<.01, and so the null hypothesis is rejected.\n\nThe numbered images correspond to the images that are measured, and also to the figures that are illustrated in this article.\n\nThe next null hypothesis tested, based on the data for the TEM of PD tissue and of controls was that the positive TEM result is independent of whether the cases are of PD or else controls. Using a chi-squared test for categorical data, the test statistic was calculated at 16.8. With one degree of freedom, the null hypothesis was rejected at the 1% significance level. Using the Fisher exact test, the hypergeometric probability of the results being obtained if the null hypothesis holds is 1.3 × 10-4. The null hypothesis is thus again rejected.\n\nThe third null hypothesis tested was that the observed positive immunohistochemical staining of sections using anti-coxsackie antibody or anti-poliovirus antibody is independent of whether the cases are of PD or else controls. Again using a chi-squared test for categorical data, the test statistic was calculated at 11.4 for anti-coxsackie antibody, which rejects the null hypothesis at the 1% significance level; and 6.3 for anti-poliovirus antibody, which does not reject the null hypothesis at the 1% significance level but does at 5%. Using the Fisher exact test, the hypergeometric probability of the anti-coxsackie antibody results being obtained if the null hypothesis holds is 8.1 × 10-4, so that the null hypothesis is again rejected at 1% significance level. For the anti-poliovirus antibody, the hypergeometric probability is calculated at 0.014, so the null hypothesis cannot be rejected at the 1% significance level, but is rejected at 5%.\n\n\nDiscussion and conclusions\n\nIn a previous communication, we reported the following TEM finding: the assembly in the nuclei and cytoplasm of polio virus particles and coxsackie virus particles that measured from 20 nm to 40 nm in virus factories of virus infected cell cultures1. The finding of virus particles in the nuclei of cells infected with an RNA virus was a novel observation. Virus factory has been described as a specific intracellular compartment where viral components concentrate by Novoa et al.27. Virus factories often include membrane components that are involved in virus replication. This has been demonstrated in our PD study (Figure 3 and Figure 5). The measurements we made by TEM of poliovirus in infected cells are in contrast to previous reports on the diameter of poliovirus virions using cryo-electron microscopy of purified virus preparations. In the latter studies the diameter of the virions was 30 nm. Larger diameter particles were not reported. This may be due to selection in the purification procedure of the population of virions for study28,29.\n\nThe results of TEM of PD brain are comparable to the TEM finding of virus particles found in cell cultures infected with poliovirus and coxsackie virus1. The VLP found in PD brain are also comparable to the VLP in brain from encephalitis lethargica patients1, and the virus particles found in this study in human spinal cord neurons in cases of human poliomyelitis. We had confirmed the finding of enterovirus in encephalitis lethargica brain by the demonstration of a partial enterovirus nucleotide sequence in brain tissue1. In the present study, the observation that the VLP in many of the cytoplasmic virus factories in the PD cases are larger than the cytoplasmic ribosomes from control cases was confirmed by statistical analysis (Table 1 and Table 4). In certain PD cases, the cytoplasmic factories consisted of incomplete VLP measuring approximately 20 nm. There was also a membrane component to the virus factories (Figure 3 and Figure 5). We observed that similar cytoplasmic virus factories were in the human spinal cord neurons infected with polio virus (Figure 8–Figure 10). These findings suggest that the VLP in PD represent an enterovirus infection. Intranuclear VLP, measuring approximately 40 nm, were observed in the nuclei of motor neurons in cases of poliomyelitis (Figure 11).\n\nBy analogy to recent studies on Alzheimer’s disease14,15, we suggest that an enterovirus infection in PD may act as a seed for the replication of misfolded α-synuclein protein in addition to the direct cytopathic effect of a virus infection on neurons.\n\nPrevious studies did not consider the possibility of enterovirus infection in PD in experiments conducted to isolate virus11–15. We propose that experiments to isolate enterovirus from PD brain be carried out using PD tissue co-cultivated with cell cultures that carry membrane receptors for enteroviruses. Sequence analysis may be carried out using either virus isolated by cell culture or directly from PD brain tissue using an enterovirus sequence primer. The partial nucleotide sequence of encephalitis lethargica may be employed1.\n\n\nEthical statement\n\nStudy of brain specimens had been cleared for ethical agreement by the National Research Ethics Committee for Oxfordshire UK, rec no.: 07/H0606/85. The brain samples from human autopsy material were obtained from the pathology collections of the John Radcliffe Hospital, Oxford UK, and from the former Armed Forces Institute of Pathology, Washington DC, USA. These institutions approved the use of the tissue for research, and they were satisfied that no further ethical approval was required. In the case of the material from the UK, the principal author holds the “Release of Tissue Disclaimer” from the Thomas Willis Oxford Collection, at the Neuropathology Department John Radcliffe Hospital, Oxford.\n\n\nData availability\n\nTable 2 and Table 3 report the detailed IHC results for IHC tests.\n\nThe TEM images presented here are listed in the legends by the author’s TEM serial number followed by the donor’s autopsy number. Copies of the images are stored at the Public Health England electron microscopy laboratory. They may be freely obtained by contacting Dr. Matthew Hannah at PHE.", "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\nMs. Glynnis Dunn at the Department of Virology at the National Institute for Biological Standards and Control donated the antisera for immunohistochemical use. Professor Margaret Esiri at the Department of Neuropathology, JRH, Oxford UK (retired), contributed brain tissue from the Oxford Brain Bank from PD and control cases. She gave advice concerning the TEM and IHC results. Professor David Ferguson at JRH gave access to the electron microscope at JRH. Professor David Brown at Public Health England arranged for the use of the electron microscope. Professor Alex Henneberg (Frankfurt) gave nasal and colonic biopsies from PD patients for TEM study. Mr. Peter Locker gave help with computing. Mrs. Sydnee Dourmashkin gave personal support.\n\n\nReferences\n\nDourmashkin RR, Dunn G, Castano V, et al.: Evidence for an enterovirus as the cause of encephalitis lethargica. BMC Infect Dis. 2012; 12(6): 136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParkinson J: An essay on the shaking palsy. Reprinted by Dawsons of Pall Mall, 16 Pall Mall, London SW 1. 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PubMed Abstract\n\nHawkes CH, Del Tridici K, Braak H: Parkinson's disease: the dual hit theory revisited. Ann N Y Acad Sci. 2009; 1170(7): 615–22. PubMed Abstract \| Publisher Full Text\n\nSvensson E, Horváth-Puhó E, Thomsen RW, et al.: Vagotomy and subsequent risk of Parkinson’s disease. Ann Neurol. 2015; 78(4): 522–9. PubMed Abstract \| Publisher Full Text\n\nLiu B, Fang F, Pedersen NL, et al.: Vagotomy and Parkinson disease: A Swedish register-based matched-cohort study. Neurology. 2017; 88(21): 1996–2002. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGibbs CJ Jr, Gajdusek DC: An update on long-term in vivo and in vitro studies designed to identify a virus as the cause of amyotrophic lateral sclerosis, Parkinsonism dementia, and Parkinson’s disease. Adv Neurol. 1982; 36: 343–353. PubMed Abstract\n\nWetmur JG, Schwartz J, Elizan TS: Nucleic acid homology studies of viral nucleic acids in idiopathic Parkinson’s disease. Arch Neurol. 1979; 36(8): 462–464. PubMed Abstract \| Publisher Full Text\n\nElizan TS, Schwartz J, Yahr MD: Antibodies against arboviruses in Postencephalitic and idiopathic Parkinson’s disease. Arch Neurol. 1978; 35(5): 257–260. PubMed Abstract \| Publisher Full Text\n\nElizan TS, Madden DL, Noble GR, et al.: Viral antibodies in serum and CSF of Parkinsonian patients and controls. Arch Neurol. 1979; 36(9): 529–534. PubMed Abstract \| Publisher Full Text\n\nSchwartz J, Elizan TS: Search for viral particles and virus-specific products in idiopathic Parkinson disease brain material. Ann Neurol. 1979; 6(3): 261–3. PubMed Abstract \| Publisher Full Text\n\nJaunmuktane Z, Mead S, Ellis M, et al.: Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy. Nature. 2015; 525(7568): 247–50. PubMed Abstract \| Publisher Full Text\n\nJucker M, Walker LC: Neurodegeneration: Amyloid-β pathology induced in humans. Nature. 2015; 525(10): 193–4. PubMed Abstract \| Publisher Full Text\n\nSampson TR, Debelius JW, Thron T, et al.: Gut microbiota regulate motor deficits and neuroinflammation in a model of Parkinson’s Disease. Cell. 2016; 167(6): 1469–1480.e12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavie CA: A review of Parkinson’s disease. Br Med Bull. 2008; 86(1): 109–127. PubMed Abstract \| Publisher Full Text\n\nMok KY, Sheerin U, Simon-Sanchez J, et al.: Deletions at 22q11.2 in idiopathic Parkinson’s disease: a combined analysis of genome-wide association data. Lancet Neurol. 2016; 15(6): 585–596. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHerva ME, Spillantini MG: Parkinson’s disease as a member of Prion-like disorders. Virus Res. 2015; 207(9): 38–46. PubMed Abstract \| Publisher Full Text\n\nBarker RA, Williams-Grey CH: Review: The spectrum of clinical features seen with alpha synuclein pathology. Neuropathol Appl Neurobiol. 2016; 42(1): 6–19. PubMed Abstract \| Publisher Full Text\n\nJosephs KA, Parisi JE: Alpha-synuclein studies are negative in postencephalic parkinsonism of von Economo. Neurology. 2002; 59(4): 545–646. PubMed Abstract \| Publisher Full Text\n\nMunhoz R, Filho DB, Teive HA: Not all drug-induced parkinsonism are the same: the effect of drug class on motor phenotype. Neurol Sci. 2017; 38(2): 319–324. PubMed Abstract \| Publisher Full Text\n\nCrystal SC, Leonidas J, Jakubowski A, et al.: Thalidomide induced acute worsening of Parkinson’s disease. Mov Disord. 2009; 24(12): 1863–4. PubMed Abstract \| Publisher Full Text\n\nVector Laboratories. 3 Accent Park, Bakewell Road, Orton, Southgate, Peterborough, PE2 6XS, UK. Reference Source\n\nNovoa RR, Calderita G, Arranz R, et al.: Virus factories: associations of cell organelles for viral replication and morphogenesis. Biol Cell. 2005; 97(2): 147–72. PubMed Abstract \| Publisher Full Text\n\nBubeck D, Filman DJ, Cheng N, et al.: The structure of the poliovirus 135S cell entry intermediate at 10-angstrom resolution reveals the location of an externalized polypeptide that binds to membranes. J Virol. 2005; 79(12): 7745–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nButan C, Filman DJ, Hogle M: Cryo-electron microscopy reconstruction shows poliovirus 135S particles poised for membrane Interaction and RNA release. J Virol. 2014; 88(3): 1758–70. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "31767", "date": "21 Mar 2018", "name": "Steven Patterson", "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\n\nUltrastructural and immunohistochemical analysis of brain tissue from Parkinson’s disease patients could provide important data on the pathogenesis of the disease. Electron microscopy of autopsy tissue, particularly after formalin fixation and embedding for light microscopy, is challenging as preservation is far from optimal. Despite this difficulty the authors have provided persuasive evidence of cytoplasmic structures that may be viral. However, although the authors have convincingly shown differences between Parkinson’s and control brain tissue I feel they should be more cautious in their rather definitive interpretation that there are enterovirus structures antigenically related to polio and coxsachie virus present. In addition I think the discussion should be expanded as indicated below.\n\nMajor specific comments\nThe ICH showed labelling for both coxsachie and polio virus antigen. What evidence is there in the literature that these viruses have common antigens which may explain their findings? In their studies was either pre-immune or non-immune serum used as a control? Does binding of the coxsachie antiserum block subsequent labelling with the polio virus antiserum and vice versa?\n\nThe micrographs of the cytoplasmic virus factories in the Parkinson’s tissue are persuasive of enterovirus infection, particularly in view of the similarity to cytoplasmic structures in polio brain tissue and the absence in controls. However, the presence of nuclear virus particles would not be expected of an enterovirus infection. This observation should be more extensively discussed. In addition the extensive early em studies in the 1960s by authors such as Dales (Dales et al 1965 Virology 26, 389)\n\nshould be cited.\n\nIs it possible to use haemotoxylin to stain nuclei in the IHC to confirm or not the presence of nuclear viral antigen?\n\nTable 3 summarises the em and IHC data showing that for 13 samples there was both em and IHC. All 13 were positive for virus factories by em but 8/13 and 7/13 were positive for staining with coxsachie and poliovirus antiserum respectively. Given that it may be expected that IHC is more sensitive since a larger area of tissue may be examined the authors should comment on the difference between the em and IHC findings.\nMinor comments\n\nI presume that the tissue was fixed in formalin, this should be stated in methods\n\nFor the reader it would be helpful if a micrograph could be shown in which both ribosomes and VLPs are indicated\n\nFigure 7 legend states that an arrow indicates the nuclear membrane, I cannot see an arrow on this micrograph.\n\nThere appears to be a core in one of the nuclear particles shown in figure 11, indicated by the arrow, can the authors comment.\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? Partly", "responses": [ { "c_id": "3643", "date": "05 Jun 2018", "name": "Steven Patterson", "role": "Reviewer Response", "response": "The amendments made by the authors have satisfied my concerns. Steven Patterson" } ] }, { "id": "31751", "date": "03 Apr 2018", "name": "Doris Bucher", "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\nComments/minor revisions\nRef. 16 refers to Treatment of children with human cadaver-derived growth hormone (hGH) contaminated with prions resulted in iatrogenic transmission of CreutzfeldtJakob disease (iCJD). I was unable to find any reference to ‘immature dopamine neurons grafted to PD patients” and Lewy bodies in Ref. 16. Need correct reference or further explanation.\n\nFig. 4 - would be helpful to use arrows to indicate several VLPs which were used in measurements.\n\nFig. 7 - need arrow - VLP In a PD neuron, close to the internal face of the nuclear membrane.\n\nRe: Immunohistochemistry Methods: Was antiserum raised against polio types 1, 2, and 3 or…only poliovirus type 1?\n\nFig. 11 - How were these VLP particles identified as intranuclear? Are smaller particles also seen - i.e. the cluster of particles in the quadrant directly above the putative VLP?\n\nFig. 12 - what are the particles seen inside the Lewy body?\n\nFig. 16 - Shows only IHC for rabbit anti-polio. Would be useful to show an example of IHC for anti-coxsackie.\n\nTable 3 - IHC for anti-coxsackie and anti-polio - nearly all dual positives, only one is specific for coxsackie only.Is this result expected?\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/7-302
https://f1000research.com/articles/6-1642/v1	05 Sep 17	{ "type": "Research Article", "title": "The unhappy postdoc: a survey based study", "authors": [ "Amir Grinstein", "Roi Treister", "Roi Treister" ], "abstract": "Background: The emerging public discourse about the “broken” postdoc system is mostly conceptual. The current work offers an attempt to quantify postdocs’ perceptions, goals, and well-being. Methods: A survey of 190 postdocs in North America. Results: This article first reveals a surprisingly unhappy postdoc community with low satisfaction with life scores. Second, it demonstrates how over the course of the fellowship many postdocs lose interest in the goal of pursuing a tenure track academic position (~20%) or in recommending the postdoc track to others (~30%). Finally, we find that among a large number of factors that can enhance satisfaction with life for postdocs (e.g., publication productivity, resources available to them) only one factor stood out as significant: the degree to which atmosphere in the lab is pleasant and collegial. Conclusions: Our findings can stimulate policy, managerial, and career development improvements in the context of the postdoc system.", "keywords": [ "Postdoc", "post-doctorate", "well-being", "academic career" ], "content": "Introduction\n\nPost-doctorate fellows (i.e. postdocs) are a major force in advancing scientific research, and often are the driving force behind successful labs, especially in the bio-medical area. Not only the sheer number of postdocs is on the rise – the number of postdocs has tripled since 1979 (Gould, 2015) – but also their research projects require heavier funding (Davis, 2009; Xuhong, 2013). A common assumption is that PhDs pursue a postdoc position in an academic research institution to enhance their research skills and reputation, which in turn increase their chances of obtaining the ultimate goal: a tenure track academic appointment. While this is a worthy goal to pursue, and there is no doubt that a postdoc position is often key for a future academic appointment, there are growing concerns that the postdoc system is broken and unsustainable (Alberts et al., 2014; Gould, 2015). Such concerns are mostly heard from postdocs (Powell, 2015; Smaglik, 2016). Conversely, the academic establishment benefits from maintaining the status quo because the supply of skilled employees like postdocs – that are relatively non-costly and require minimal training and supervision – is highly warranted (Smaglik, 2016). However, the benefits from a postdoc career for the postdocs themselves are becoming much less evident. Under unstable economic conditions tenure track academic appointments become tremendously difficult to obtain. Recent evidence from the UK, for example, suggests that of 100 science PhD graduates, about 30 will go on to postdoc research, but just 4 will secure permanent academic posts with a significant research component (Nature editorial, 2014). In the US, the situation is slightly better: 65% of all PhD holders follow the postdoc path but only 15–20% of those gain a tenure track position (Gould, 2015). Moreover, postdocs that are not able to achieve an academic appointment often become over-qualified for industry positions while losing alternative higher compensation (salaries in the industry) and often putting their personal life (marriage, kids) on hold.\n\nIt takes between 12–18 years of academic training to get into the entry level of an academic tenure track position (Gould, 2015). This very long training process has economic, social, and individual well-being costs. These heavy costs are often complemented with the significant uncertainty surrounding the likelihood of obtaining a tenure track academic appointment. Overall therefore, it might be very useful for potential postdocs to develop a more critical view of the traditional academic postdoc track and of their career choices after completing their PhD.\n\nWhile discussions of the postdoc reality have been attracting growing attention (e.g., Alberts et al., 2014; Gould, 2013; Powell, 2015; Smaglik, 2016), they have mostly focused on the policy level and lacked empirical assessments at the individual postdoc level. Our aim is to empirically study the perspective of postdocs, to better understand their goals and perceptions, and to be able to promote evidence-based career choices by PhDs considering the postdoc path. We follow recent efforts such as the postdocs survey conducted by Gibbs et al. (2015). Specifically, we pose, at the individual level, the following unanswered research questions:\n\nGiven the complex reality postdocs face today\n\n(1) How satisfied are current postdocs?\n\n(2) How likely are they to change – over the course of their fellowship – their key career goal of (typically) obtaining a tenure track appointment?\n\n\nMethods\n\nTo answer our research questions, we conducted a survey of postdocs in North America, mostly in the bio-medical and physical sciences. We emailed the survey to 29 leading postdoc associations in North America (e.g., National Postdocs Association, Rockefeller Postdocs Association, Johns Hopkins Postdoctoral Association) asking them to distribute it among their members. Overall we generated responses from 190 postdocs1. Respondents’ anonymity was kept. The majority of respondents were positioned in the U.S, with 6 participants from Europe, Asia and Africa2. Table 1 summarizes key characteristics of the surveyed postdocs. The survey, data, and list of postdoc associations targeted can be found as a supplementary files (Dataset 1, Supplementary file 1, Supplementary file 2).\n\nDifferent n are due to missing values. Percentages are of valid cases.\n\nIn the survey we were especially interested in collecting data regarding satisfaction levels of postdocs as well as their career goals dynamics while accounting for variety of factors that may impact these outcomes such as number of publications and atmosphere in the lab.\n\n\nResults\n\nA first set of findings suggests that postdocs are far from being satisfied with their current situation in life with a mean of 4.47 (SD=1.46). Satisfaction was quantified by five items, each reported on a 1–7 scale, based on the established Diener et al.’s (1985) scale; α=.90: “In most ways my life is close to my ideal,” “The conditions of my life are excellent,” “I am satisfied with my life,” “So far I have gotten the important things I want in life,” “If I could live my life over, I would change almost nothing”. Further, 30% of participants demonstrate lower than the median (=4) satisfaction levels. Considering that the established research using the same satisfaction scale typically suggests a positive bias of people when asked about satisfaction with life (Abdallah et al., 2009; Diener et al., 1985), our results demonstrate a surprisingly low well-being among people that are one step away from their “dream” appointment position and are typically already affiliated with a top-tier academic affiliation. Further, prior work on satisfaction with life of other type of skilled trainees that are roughly at the same age group (e.g., medical students) report much higher satisfaction levels (not lower than 5.2 (e.g., Kjeldstadli et al., 2006; Samaranayake & Fernando, 2011). Although a less “clean” comparison, it is surprising to find that the postdocs in our sample are less content than people in many poor, underdeveloped countries according to a comparable analysis of people’s satisfaction with life across countries (Figure 1; adapted from Abdallah et al., 2009)3. Further, the postdocs fall significantly behind the general US population, where many postdocs reside.\n\nAdapted from Abdallah et al. (2009).\n\nInterestingly, out of a list of potential explanatory variables of satisfaction with life among postdocs – including postdocs’ demographic characteristics (age, gender), personal characteristics (number of postdocs, number of publications, years in postdoc, discipline), publication productivity (number of publications, number of publications as a first author, number of publications based on their postdoc as first authors or in general), PI characteristics (number of publications, frequent interaction of the postdocs with the PI), and lab characteristics (value of equipment, number of postdocs) – only one factor showed significant relationship with satisfaction with life: atmosphere in the lab (r=.247, p=.002). This measure included five items on a 1–7 scale, inspired by Moos’ (2008) Work Environment Scale’s Peer Cohesion sub-dimension aiming to understand how friendly and supportive employees are to each other (α=.80 “The atmosphere in the lab is/was very pleasant,” “I am/was happy to go to work in the morning,” “I view/viewed my lab colleagues as friends,” “I often have/had social interactions with my lab colleagues outside the lab,” “I often collaborate/collaborated on joint projects with my lab colleagues”).\n\nA key consequence of such lack of satisfaction is represented in participants’ responses to a question wondering whether the postdocs will likely to recommend the postdoc track to other who considering it. Only 28.4% “agreed” or “definitely agreed” to recommend the postdoc path to others. Less than a third.\n\nAnother finding is that many postdocs are re-considering or re-considered their career goals during their fellowship. We asked participants to share with us their main career goal when starting their postdoc and also at the point of the survey. Options included (a) university faculty with an emphasis on teaching, (b) university faculty with an emphasis on research, (c) government job with an emphasis on R&D, (d) job in an established firm with an emphasis on R&D, (e) job in a start-up with an emphasis on R&D or (f) other. The results (summarized in Figure 2) shows a shift from a goal focusing on academic tenure track position to other goals – mostly industry positions. In our sample, 71.3% of the postdocs began their fellowship with the goal of pursuing a tenure track academic position. When the survey was conducted only 50% maintained a similar career goal. The key career change involves a focus on the industry (established R&D company or a start up) – from 9% and 3%, to 18% and 8%, respectively.\n\nMost striking is the finding that 71.3% of the surveyed postdocs began their fellowship with the goal of pursuing a tenure track academic position but when the survey was conducted only 50% maintained this career goal.\n\n\nDiscussion\n\nMany postdocs are facing a well-being paradox. On the one hand, a postdoc position is a huge step towards achieving many postdocs’ central goal of obtaining a tenure track appointment. On the other hand, however, the growing realization that these positions are scarce than ever before, as well as the long, frustrating, and not always rewarding postdoc journey significantly damages the well-being and satisfaction of many postdocs.\n\nOur empirical analysis is a valuable first step in documenting and reflecting on the notion of the well-being paradox of postdocs. Unhappy postdocs is not a good recipe for sustainable success of the postdoc system and for advancing top-tier scientific work. This means – from the perspective of policy makers, university administration, and lab leaders – that there is value in better understanding and catering to the well-being and needs of individual postdocs. Our finding that a key aspect of postdocs’ satisfaction with life involves a positive atmosphere in the lab attest to the importance of “soft”, not-science related factors that are essentials to the sustainability of a successful postdoc system. Such insight can help lab leaders in postdoc recruitment efforts and in optimizing the postdoc experience.\n\nFurther, from the perspective of postdocs and prospective postdocs (mostly PhDs), they should consider adopting a more critical view of the traditional academic postdoc track. This may lead to seriously considering all available career options including an industry position following the PhD, an industry postdoc, or a combined academic-industry postdoc. A recent finding that even after achieving a tenure-track academic position, many assistant professors are unhappy (Nature editorial, 2016), could be another catalysts for postdocs to re-think their career goals. Overall, these trends may require the industry to be more proactive in establishing postdoc positions while requiring the academic system to be more open and flexible with respect to non-academic postdocs and collaboration with the industry.\n\nOverall, our work helps to better understand the well-being of postdocs and its drivers. It provides empirical support to the idea that the current postdoc system is broken and that postdocs are paying a price in well-being terms. Any successful change in the postdoc system would need to enhance postdocs’ well-being and it is our hope that our findings stimulate policy, managerial, and career development improvements that can be pursued.\n\n\nData availability\n\nDataset 1\n\nSurvey Response: A dataset including the response of 190 North American postdocs. 10.5256/f1000research.12538.d176428 (Grinstein & Treister, 2017)\n\n\nEthics and consent\n\nThe first author’s university ethics committee (Northeastern University’s Institutional Review Board) has approved the project “The Unhappy Postdoc” (IRB#: 15-05-01). Written informed consent was obtained from all participants.\n\n\nNotes\n\n1Unfortunately we did not get access to the associations’ member lists or to information about them so we lack data on the overall number of survey invitations sent.\n\n2These 6 postdocs where part of the associations and have recently moved back to their home country.\n\n3To be compatible with Abdallah et al.’s (2009) map, we transformed our 1–7 satisfaction with life scale to a 1–10 scale, on which the surveyed postdocs demonstrate a 6.1 satisfaction with life score.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe study was self-funded.\n\n\nAcknowledgment\n\nWe are thankful for a postdoc that helped us with crafting the idea and executing the research but decided to remain anonymous due to the potential negative consequences arising from a study highlighting the “broken system” s/he is part of.\n\n\nSupplementary Material\n\nSupplementary file 1: A survey that was distributed among 190 North American postdocs.\n\nClick here to access the data.\n\nSupplementary file 2: List of postdoc associations targeted legend: a list of 29 central postdoc associations in North America that helped distribute the survey among their members.\n\nClick here to access the data.\n\n\nReferences\n\nAbdallah S, Thompson S, Michaelson J, et al.: The Happy Planet Index 2.0: Why Good Lives Don’t Have to Cost the Earth. London: nef (the new economics foundation). 2009. Reference Source\n\nAlberts B, Kirschner MW, Tilghman S, et al.: Rescuing US biomedical research from its systemic flaws. Proc Natl Acad Sci U S A. 2014; 111(16): 5773–5777. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavis G: Improving the Postdoctoral Experience: An Empirical Approach. In “Science and Engineering Careers in the United States: An Analysis of Markets and Employment”. R. B. Freeman and D. L. Goroff, editors, NBER, Chapter URL:http://www.nber.org/chapters/c11619. 2009; 99–127. Publisher Full Text\n\nDiener E, Emmons RA, Larsen RJ, et al.: The Satisfaction With Life Scale. J Pers Assess. 1985; 49(1): 71–75. PubMed Abstract \| Publisher Full Text\n\nEditorial: Harsh reality. Nature. 2014; 516(7529): 7–8. PubMed Abstract \| Publisher Full Text\n\nEditorial: Young researchers thrive in life after academia. Nature. 2016; 537(7622): 585. PubMed Abstract \| Publisher Full Text\n\nGibbs KD Jr, McGready J, Griffin K: Career Development among American Biomedical Postdocs. CBE Life Sci Educ. 2015; 14(4): ar44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGould J: How to build a better PhD. Nature. 2015; 528(7580): 22–25. PubMed Abstract \| Publisher Full Text\n\nGrinstein A, Treister R: Dataset 1 in: The unhappy postdoc: a survey based study. F1000Research. 2017. Data Source\n\nKjeldstadli K, Tyssen R, Finset A, et al.: Life satisfaction and resilience in medical school--a six-year longitudinal, nationwide and comparative study. BMC Med Educ. 2006; 6(1): 48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoos R: Work Environment Scale Manual. (4th ed.). Palo Alto, CA: Mind Garden, Inc. 2008. Reference Source\n\nPowell K: The future of the postdoc. Nature. 2015; 520(7546): 144–147. PubMed Abstract \| Publisher Full Text\n\nSamaranayake CB, Fernando AT: Satisfaction with Life and Depression among Medical Students in Auckland, New Zealand. N Z Med J. 2011; 124(1341): 12–17. PubMed Abstract\n\nSmaglik P: Activism: Frustrated postdocs rise up. Nature. 2016; 530(7591): 505–506. PubMed Abstract \| Publisher Full Text\n\nXuhong S: The Impacts of Postdoctoral Training on Scientists’ Academic Employment. J Higher Educ. 2013; 84(2): 239–265. Publisher Full Text" }	[ { "id": "26232", "date": "09 Oct 2017", "name": "Navid Ghaffarzadegan", "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\nOverall this is an interesting work and it is well-written. The study explores satisfaction with life of postdocs. It also looks at career goal change of postdocs. The study contributes to our understanding of the current mental state of postdocs, a growing sub-population of the science workforce.\nThe work is clearly and accurately presented. However, there are many other current literature on this topic that authors may benefit from reading them and linking their literature review to those studies or comparing and contrasting their findings.\nThe study design is appropriate. There are two concerns: first, as the authors acknowledge the response rate was not available. Second, biomedical postdocs in this data seem to be over-represented when compared to the national sample in Powell (2015)1. As reported in other studies2-3, the chance of landing tenure-track positions for PhDs of biomedical sciences and engineering is relatively small, which may affect their satisfaction.\n\nMost of the analysis are descriptive, which is fine. The interpretations are mostly accurate. The relationship between lab atmosphere and satisfaction is interesting. However, one might worried it’s confounded by individual characteristics such as personality – that is, people with more extraversion or agreeableness will score high on both satisfaction and subjective lab atmosphere. Furthermore, a more interesting/appropriate analysis for figure 2 might be to present results for subgroups by duration of the postdoc position. Most of the source data underlying the results are available to ensure full reproducibility. It seems that the link for Supplementary file 2 leads to the same source as for Supplementary file 1 which can be fixed.\nFinally, most of the conclusions are adequately supported by the results. The only problem was the suggestion of focusing on soft (non-science) factors of the lab. While the point makes sense, since the paper is descriptive, the authors should avoid causal interpretations.\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?\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": "3616", "date": "02 May 2018", "name": "Amir Grinstein", "role": "Author Response", "response": "Referee 4 Navid Ghaffarzadegan, Grado Department of Industrial & Systems Engineering (ISE), Virginia Tech, Blacksburg, VA, USA Ran Xu, Virginia Tech, Blacksburg, VA, USA Approved with Reservations Overall this is an interesting work and it is well-written. The study explores satisfaction with life of postdocs. It also looks at career goal change of postdocs. The study contributes to our understanding of the current mental state of postdocs, a growing sub-population of the science workforce. The work is clearly and accurately presented. However, there are many other current literature on this topic that authors may benefit from reading them and linking their literature review to those studies or comparing and contrasting their findings. Reply: Thank you for this suggestion. We have followed the review team recommendation and update our literature review. The study design is appropriate. There are two concerns: first, as the authors acknowledge the response rate was not available. Second, biomedical postdocs in this data seem to be over-represented when compared to the national sample in Powell (2015)1. As reported in other studies2-3, the chance of landing tenure-track positions for PhDs of biomedical sciences and engineering is relatively small, which may affect their satisfaction. Reply: Thank you for this feedback which we now acknowledge as a potential limitation of our work. Most of the analysis are descriptive, which is fine. The interpretations are mostly accurate. The relationship between lab atmosphere and satisfaction is interesting. However, one might worried it’s confounded by individual characteristics such as personality – that is, people with more extraversion or agreeableness will score high on both satisfaction and subjective lab atmosphere. Furthermore, a more interesting/appropriate analysis for figure 2 might be to present results for subgroups by duration of the postdoc position. Reply: To account for multiple factors and provide a more rigorous testing of the data we now report a regression analysis. Most of the source data underlying the results are available to ensure full reproducibility. It seems that the link for Supplementary file 2 leads to the same source as for Supplementary file 1 which can be fixed. Reply: We corrected for this. Finally, most of the conclusions are adequately supported by the results. The only problem was the suggestion of focusing on soft (non-science) factors of the lab. While the point makes sense, since the paper is descriptive, the authors should avoid causal interpretations. Reply: We agree our finding is correlational in nature. The language we used is hopefully not too strong (e.g., the finding can “help lab leaders”) and we also now incorporate additional support from previous work that found relatively similar effects. References 1. Powell K: The future of the postdoc.Nature. 2015; 520 (7546): 144-7 PubMed Abstract \| Publisher Full Text 2. Larson RC, Ghaffarzadegan N, Xue Y: Too Many PhD Graduates or Too Few Academic Job Openings: The Basic Reproductive Number R0 in Academia.Syst Res Behav Sci. 31 (6): 745-750 PubMed Abstract \| Publisher Full Text 3. Ghaffarzadegan N, Hawley J, Larson R, Xue Y: A Note on PhD Population Growth in Biomedical Sciences.Syst Res Behav Sci. 23 (3): 402-405 PubMed Abstract \| Publisher Full Text" } ] }, { "id": "26676", "date": "11 Oct 2017", "name": "Nick Riddiford", "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\nGrinstein and Treister present and discuss survey data collected from 190 US-based postdoctoral researchers examining life satisfaction and career goals of respondents. Their data suggest that postdocs in the US are less satisfied with their life than the general population, and that their career goals drift further away from academic research as they progress though their fellowship(s). Such data is useful and adds to a growing body of evidence that point to systemic failures in the way postdocs are treated.\n\nWhile Grinstein and Treister do a good job of describing the data, the analysis is fairly light, and several questions are left unconsidered. Most importantly, in looking for correlates of life satisfaction, what does it say that the explanatory variables you have used don’t explain this data? In addition to several major revisions, I also suggest that the authors discuss how their work relates to similar data from recent articles (for example [ref1]-4.\n\nMajor revisions\nConsidering the general tone of the manuscript is descriptive, I would strongly urge plotting each of the correlation analyses (life satisfaction vs gender, age …), and reporting R2 values (I think that these are easily generated as the tests have already been done) and simply discussing any observed correlations. The lack of significance in such a small dataset does not automatically indicate that there is nothing interesting going on. At the very least, I think the correlation between “lab atmosphere” and life satisfaction should be plotted, and the tests/software used for the correlation analyses clearly detailed in the methods section (linear regression?). Furthermore, a correlation coefficient of 0.247 is reported – is this really the R value or the R2? If the former, then the R2 = 0.06, which means that “lab atmosphere” explains only 6% of the variability in reported life satisfaction scores, which suggests there could be some other explanatory variables that haven’t been considered. This should be discussed.\n\nA second major issue relates to Figure 1. Here, it is reported in the first sentence of the results that the mean satisfaction score was 4.47, but the colouring of figure 1 suggests that postdocs fall in the 5.5-7.0 bracket. In either case, I would suggest that this only serves to highlight the many caveats of a) self-reporting of life satisfaction and b) making comparisons between data sets collected under different conditions. Do the authors really think that, with a mean life satisfaction of 4.47, postdocs in the US are (quite considerably) less satisfied with life than residents of some of the poorest, most poverty-stricken countries in the world? If not, I recommend that this figure be removed entirely. Perhaps a more meaningful comparison to make would be between the US population (per state/demographic?) and US-based postdocs. To ensure reproducibility, Dataset 1 should be resubmitted as an excel compatible file (or.txt, .csv) instead of, or in addition to, the .sav they currently provide.\n\nMinor points\nWhat do the alpha values refer to throughout the manuscript (e.g. α=.90)? These either need to be removed or discussed in the Methods section. Table 1 should be more formally presented (or see suggestion 1) and formatted i.e succinct, descriptive title and footnote describing ‘n’, and why there are missing values i.e discrepancy between 190 and 178). This applies to all other figures: The title should be concise, and followed by a legend that describes the figure in sufficient detail. Commentaries on the figure – such as in Fig.2 – belong in the main body of the results section. Why do you make the assumption that postdocs who answered your survey are “typically already affiliated with a top-tier academic affiliation”? “Satisfaction with life” should be replaced with “life satisfaction” throughout manuscript. Remove “Less than a third” in sentence containing “Only 28.4% “agreed” or “definitely agreed” to recommend the postdoc path to others. Less than a third.”. Figure 2 should be reproduced at a higher quality – it appears very pixilated on my screen.\n\nGrammatical mistakes Introduction\nReplace “kids” with “children” or similar in sentence containing “personal life (marriage, kids) on hold.”\nResults\nReplace “Further” with “Furthermore” in sentence containing “Further, 30% of participants demonstrate” Consider revising repetitive sentence containing “typically already affiliated with a top-tier academic affiliation” Pluralise “type” in sentence containing “Further, prior work on satisfaction with life of other type of skilled trainees”\nDiscussion\nReplace “scarce” with “scarcer” or “more scarce” in sentence containing “On the other hand, however, the growing realization that these positions are scarce than ever before” Remove “they” from sentence containing “they should consider adopting” Catalysts should not be plural in sentence containing “could be another catalysts for postdocs to re-think”\n\nSeveral recent survey-based articles that would be worth comparing to\nReader’s responses from Nature poll1 Analysis of academic workforce in US3 Large analysis of US census data2 Survey of UK-based biomedical researchers4 The Royal Society: The Scientific Century: securing our future prosperity. 2010\n\nSuggestions\nTable 1 would be better represented as a several bar charts You report that “out of a list of potential explanatory variables of satisfaction with life among postdocs … only one factor …”. Even if this is mostly negative data, this would make a nice figure, as it’s quite surprising to me that you find no correlation between some of your explanatory variables Representing Fig.2 as a 3D bar chart adds nothing at a loss of clarity. Consider replotting as a conventional 2D bar chart\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?\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? Yes", "responses": [ { "c_id": "3615", "date": "02 May 2018", "name": "Amir Grinstein", "role": "Author Response", "response": "Referee 3 Nick Riddiford, Institut Curie, Paris, France Approved with Reservations Grinstein and Treister present and discuss survey data collected from 190 US-based postdoctoral researchers examining life satisfaction and career goals of respondents. Their data suggest that postdocs in the US are less satisfied with their life than the general population, and that their career goals drift further away from academic research as they progress though their fellowship(s). Such data is useful and adds to a growing body of evidence that point to systemic failures in the way postdocs are treated. While Grinstein and Treister do a good job of describing the data, the analysis is fairly light, and several questions are left unconsidered. Most importantly, in looking for correlates of life satisfaction, what does it say that the explanatory variables you have used don’t explain this data? In addition to several major revisions, I also suggest that the authors discuss how their work relates to similar data from recent articles (for example [ref1]-4. Major revisions Considering the general tone of the manuscript is descriptive, I would strongly urge plotting each of the correlation analyses (life satisfaction vs gender, age …), and reporting R2 values (I think that these are easily generated as the tests have already been done) and simply discussing any observed correlations. The lack of significance in such a small dataset does not automatically indicate that there is nothing interesting going on. At the very least, I think the correlation between “lab atmosphere” and life satisfaction should be plotted, and the tests/software used for the correlation analyses clearly detailed in the methods section (linear regression?). Furthermore, a correlation coefficient of 0.247 is reported – is this really the R value or the R2? If the former, then the R2 = 0.06, which means that “lab atmosphere” explains only 6% of the variability in reported life satisfaction scores, which suggests there could be some other explanatory variables that haven’t been considered. This should be discussed. Reply: Thank you for pushing us to more rigorously test our results. We now report in more detail the correlation matrix and a more rigorous regression analysis. A second major issue relates to Figure 1. Here, it is reported in the first sentence of the results that the mean satisfaction score was 4.47, but the colouring of figure 1 suggests that postdocs fall in the 5.5-7.0 bracket. In either case, I would suggest that this only serves to highlight the many caveats of a) self-reporting of life satisfaction and b) making comparisons between data sets collected under different conditions. Do the authors really think that, with a mean life satisfaction of 4.47, postdocs in the US are (quite considerably) less satisfied with life than residents of some of the poorest, most poverty-stricken countries in the world? If not, I recommend that this figure be removed entirely. Perhaps a more meaningful comparison to make would be between the US population (per state/demographic?) and US-based postdocs. Reply: We now present our results more clearly in the context of other studies examining various populations’ life satisfaction (a good review is Diener and Pavot, 1993). The mean life satisfaction we found for postdocs is the lowest compared to all other student groups in developed countries or professionals (apart from “elderly caregivers”) as can be seen from Table 1 in Diener and Pavot (1993). We are sorry for not being clear enough about the difference between the text and the use of our scale and the Figure. The decision to adapt our scale for the purpose of the figure appeared in footnote 3 of the original version of the manuscript: “To be compatible with Abdallah et al.’s (2009) map, we transformed our 1-7 satisfaction with life scale to a 1-10 scale, on which the surveyed postdocs demonstrate a 6.1 satisfaction with life score.” Still, given the feedback from the review team we have decided to remove Figure 1 from the revised manuscript. Further, following your suggestions we toned down our discussion and removed some of the “not clean” comparisons. Noteworthy that our findings do suggest that in the context of developed countries, our results actually show a surprisingly low life satisfaction score of postdocs. We now integrate this discussion in the text. To ensure reproducibility, Dataset 1 should be resubmitted as an excel compatible file (or.txt, .csv) instead of, or in addition to, the .sav they currently provide. Reply: Done. Minor points What do the alpha values refer to throughout the manuscript (e.g. α=.90)? These either need to be removed or discussed in the Methods section. Reply: Sorry for not being clear. We now explain this better in the text (Cronbach’s Alpha that tests for the reliability of the scale). Table 1 should be more formally presented (or see suggestion 1) and formatted i.e succinct, descriptive title and footnote describing ‘n’, and why there are missing values i.e discrepancy between 190 and 178). This applies to all other figures: The title should be concise, and followed by a legend that describes the figure in sufficient detail. Commentaries on the figure – such as in Fig.2 – belong in the main body of the results section. Reply: We now include a correlation matrix in addition to the table describing participants’ profiles. We also added a table for the regression analysis results. We moved the elaborated text from Fig. 2. We kept (and better explained) some of the text below the tables that seemed the most relevant to interpret the results (coding of some variables, p values). Why do you make the assumption that postdocs who answered your survey are “typically already affiliated with a top-tier academic affiliation”? Reply: This is a fair point. We removed this unfounded assumption. “Satisfaction with life” should be replaced with “life satisfaction” throughout manuscript. Reply: Our use of “satisfaction with life” was influenced by the fact that this is the way the established measure is labeled. In the revised version we kept this term only when addressing the scale and in all other locations changed to “life satisfaction”. Remove “Less than a third” in sentence containing “Only 28.4% “agreed” or “definitely agreed” to recommend the postdoc path to others. Less than a third.”. Reply: Done. Figure 2 should be reproduced at a higher quality – it appears very pixilated on my screen. Reply: We replotted this figure as 2D bar chart and changed the colors. We hope it is clearer now. Also, please note this is now Fig. 1. Grammatical mistakes Introduction Replace “kids” with “children” or similar in sentence containing “personal life (marriage, kids) on hold.” Reply: Done. Results Replace “Further” with “Furthermore” in sentence containing “Further, 30% of participants demonstrate” Reply: Done. Consider revising repetitive sentence containing “typically already affiliated with a top-tier academic affiliation” Pluralise “type” in sentence containing “Further, prior work on satisfaction with life of other type of skilled trainees” Replace “scarce” with “scarcer” or “more scarce” in sentence containing “On the other hand, however, the growing realization that these positions are scarce than ever before” Remove “they” from sentence containing “they should consider adopting” Catalysts should not be plural in sentence containing “could be another catalysts for postdocs to re-think” Reply: We have corrected (or removed) the relevant sentences. Several recent survey-based articles that would be worth comparing to Reader’s responses from Nature poll1 Analysis of academic workforce in US3 Large analysis of US census data2 Survey of UK-based biomedical researchers4 The Royal Society: The Scientific Century: securing our future prosperity. 2010 Reply: Thank you. We have integrated some of the work that studied these sources throughout the revised manuscript. Suggestions Table 1 would be better represented as a several bar charts Reply: As we are more used to the “standard” way of reporting participants’ characteristics in a table we thought most readers would prefer a “standard” table. If this is key, we can of course make the change in a next revision effort. You report that “out of a list of potential explanatory variables of satisfaction with life among postdocs … only one factor …”. Even if this is mostly negative data, this would make a nice figure, as it’s quite surprising to me that you find no correlation between some of your explanatory variables Reply: We were not sure what type of figure would capture best this effect. In the revised version we also include a table for the regression analysis results which make results more clear and accessible. We are happy to create a figure if you have an idea about the most relevant approach. Representing Fig.2 as a 3D bar chart adds nothing at a loss of clarity. Consider replotting as a conventional 2D bar chart Reply: Done. Please note this is Fig. 1 now. References 1. Powell K: Hard work, little reward: Nature readers reveal working hours and research challenges. Nature. 2016.Publisher Full Text 2. Heggeness ML, Gunsalus KT, Pacas J, McDowell GS: Preparing for the 21st Century Biomedical Research Job Market: Using Census Data to Inform Policy and Career Decision-Making. sjscience.org. 2016. 3. Heggeness ML, Gunsalus KTW, Pacas J, McDowell G: Snapshot of the US biomedical workforce. Nature. 2016; 541: 21-23 4. Riddiford N: A survey of working conditions within biomedical research in the United Kingdom. F1000Research. 2017; 6.Publisher Full Text" } ] }, { "id": "26673", "date": "12 Oct 2017", "name": "Gary S. McDowell", "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 Unhappy Postdoc”, Grinstein and Treister present the results of a survey to 190 respondents who carried out postdoctoral training in the U.S. The results seek to demonstrate the satisfaction of postdocs with their postdoctoral training and isolate factors which are critical to their sense of well-being. So far, very few efforts have looked at the well-being of postdocs in the context of their training, and this survey data sheds some light into the murky world of the postdoc.\nThe survey has a small sample size, understandable given the difficulty of actually getting access to communicate with postdocs, but the most pressing concerns are how far selection bias plays into the inclination of postdocs to respond; and whether there was thought given to an appropriate control group? However, it is encouraging to see that some of the results replicate the findings of others, which may hint towards this being at least a fair representation of a subset of the postdoc population.\nMajor concerns:\nPlease check the proportion of US PhDs pursuing postdocs - while this is not an easy number to quantify, Kahn and Ginther 20171 and Sauermann and Roach 20162 both point to about 80% of U.S. biomedical PhDs pursuing postdocs, and the NSF’s Survey of Earned Doctorates suggests that at graduation, 30% of PhDs have a postdoc lined up, and 50% don’t know what they are doing next, suggesting a large number of people are defaulting into this plan. Again also check the tenure-track position percentage from more recent literature/qualify - it may be as low as 8% for tenure-track positions (although this may be for research-intensive institutions specifically). Sauermann and Roach 20162 should be cited - theirs is a larger survey effort of postdocs that addresses some of the same issues, particularly the change in career preference over time, and comparison with Figure 2 would be particularly useful. The first sentence in the results section would make more sense being placed after the sentence that follows it, which lays out the scale. Also the authors should explain why a mean of 4.47 and 30% of postdocs lying below the median supports their argument - what is the mean expected from other studies using the same scale? This is suggested, and one example is given, but more examples and clearer articulation of the comparison would be helpful. Also, if 30% are below the median, does that not mean 70% are at or above? It is not clear why this result is indicative of poor satisfaction and the authors should make this clearer. How does selection bias factor into the author’s analysis? One counter-argument is that the people with most to complain about felt most compelled to reply, and this skewed the results - how did the authors account for this, or is it possible to do so? Could the authors comment further on the redirection of postdocs to industry or to other non-academic careers, reflecting on the state of the current labor market? The 2012 NIH Biomedical Workforce Working Group Report found that industry is actually hiring fewer people into research positions, and into more managerial/non-research positions, and our own work looking at Census data3 show that the private sector is actually a shrinking component of the biomedical workforce, apparently explained by the increasing outsourcing of R&D to academic labs, who are both increasingly desperate for sources of funding, and also provide a plentiful pool of cheap labor. Please see also Mason et al., 20164. Reference could also be made to Faupel-Badger et al., 20175, which is possibly the only other citation that looks at career satisfaction from postdoctoral training. Can the authors link their findings to recent work that suggests that it is not the labor market, but the nature of the academic position itself, which is driving researchers out of academia? See Sauermann and Roach 20176. In Table 1, what does 'number of papers' show - is it the number published during a postdoc? In total? The survey asks this, but the table doesn't make clear which data this is. Both numbers would actually be very interesting, and also if it's possible to show how many papers people had from their first postdoc before possibly doing another one.\n\nMinor concerns:\n“Their research requires heavier funding” - could the authors articulate what they mean here? Is it that research generally has become more expensive? Or is there some increased burden at the postdoctoral position? The increasing cost of research and the market forces that are resulting in more postdocs (that they are essentially cheaper than graduate students) have been pointed out by Paula Stephan in her book “How Economics Shapes Science”7 and elsewhere, and these works should be cited appropriately in the introduction “Moreover, postdocs that are not able to achieve an academic appointment often become over-qualified for industry positions while losing alternative higher compensation (salaries in the industry)” - Kahn and Ginther 20171 should be cited here. Footnote 3 should probably be moved into the Figure Legend of Figure 1, to highlight transformation of the 1-7 scale into a 1-10 scale. In Table 1, “Duration of postdoc (years)” may be better described as “Total years in postdoctoral positions” or similar, if the survey questions are understood correctly - it is not clear whether this is pointing out the years in the current postdoc, or the years of postdoccing in total. Supplementary File 2 is not as described, but is the same as Supplementary File 1.\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": "3614", "date": "02 May 2018", "name": "Amir Grinstein", "role": "Author Response", "response": "Referee 2 Gary S McDowell, The Future of Research Inc., 848 Brockton Avenue, Abington, MA, USA; Manylabs, 1086 Folsom Street, San Francisco, CA, USA Approved with Reservations In “The Unhappy Postdoc”, Grinstein and Treister present the results of a survey to 190 respondents who carried out postdoctoral training in the U.S. The results seek to demonstrate the satisfaction of postdocs with their postdoctoral training and isolate factors which are critical to their sense of well-being. So far, very few efforts have looked at the well-being of postdocs in the context of their training, and this survey data sheds some light into the murky world of the postdoc. The survey has a small sample size, understandable given the difficulty of actually getting access to communicate with postdocs, but the most pressing concerns are how far selection bias plays into the inclination of postdocs to respond; and whether there was thought given to an appropriate control group? However, it is encouraging to see that some of the results replicate the findings of others, which may hint towards this being at least a fair representation of a subset of the postdoc population. Major concerns: Please check the proportion of US PhDs pursuing postdocs - while this is not an easy number to quantify, Kahn and Ginther 20171 and Sauermann and Roach 20162 both point to about 80% of U.S. biomedical PhDs pursuing postdocs, and the NSF’s Survey of Earned Doctorates suggests that at graduation, 30% of PhDs have a postdoc lined up, and 50% don’t know what they are doing next, suggesting a large number of people are defaulting into this plan. Again also check the tenure-track position percentage from more recent literature/qualify - it may be as low as 8% for tenure-track positions (although this may be for research-intensive institutions specifically). Reply: Thank you for these valuable sources. Based on these suggestions we updated our introduction to the manuscript. Sauermann and Roach 20162 should be cited - theirs is a larger survey effort of postdocs that addresses some of the same issues, particularly the change in career preference over time, and comparison with Figure 2 would be particularly useful. Reply: Thank you for suggesting this. This paper - and other sources suggested by the review team - are now cited and integrated into our discussion of the dynamics of postdocs’ career path. The first sentence in the results section would make more sense being placed after the sentence that follows it, which lays out the scale. Also the authors should explain why a mean of 4.47 and 30% of postdocs lying below the median supports their argument - what is the mean expected from other studies using the same scale? This is suggested, and one example is given, but more examples and clearer articulation of the comparison would be helpful. Also, if 30% are below the median, does that not mean 70% are at or above? It is not clear why this result is indicative of poor satisfaction and the authors should make this clearer. Reply: First, we made the suggested editing change. Also, we now present our results more clearly in the context of other studies examining various populations’ life satisfaction (a good review is Diener and Pavot, 1993). The mean life satisfaction we found for postdocs is the lowest compared to all other student groups in developed countries or professionals (apart from “elderly caregivers”) as can be seen from Table 1 in Diener and Pavot (1993). How does selection bias factor into the author’s analysis? One counter-argument is that the people with most to complain about felt most compelled to reply, and this skewed the results - how did the authors account for this, or is it possible to do so? Reply: This is a valid point. One way of addressing this is indeed comparing our findings to many other populations and studies – as we do now more rigorously do – with the understanding that such a potential bias can play in any surveyed group. Further, we now acknowledge this potential limitation in our GD. Could the authors comment further on the redirection of postdocs to industry or to other non-academic careers, reflecting on the state of the current labor market? The 2012 NIH Biomedical Workforce Working Group Report found that industry is actually hiring fewer people into research positions, and into more managerial/non-research positions, and our own work looking at Census data3 show that the private sector is actually a shrinking component of the biomedical workforce, apparently explained by the increasing outsourcing of R&D to academic labs, who are both increasingly desperate for sources of funding, and also provide a plentiful pool of cheap labor. Please see also Mason et al., 20164. Reply: Thank you for highlighting these evidence and research. We now integrate them to our discussion of the study’s implications. Reference could also be made to Faupel-Badger et al., 20175, which is possibly the only other citation that looks at career satisfaction from postdoctoral training. Reply: Thank you. We now cite this work. Can the authors link their findings to recent work that suggests that it is not the labor market, but the nature of the academic position itself, which is driving researchers out of academia? See Sauermann and Roach 20176. Reply: We now include this idea in our concluding discussion. In Table 1, what does 'number of papers' show - is it the number published during a postdoc? In total? The survey asks this, but the table doesn't make clear which data this is. Both numbers would actually be very interesting, and also if it's possible to show how many papers people had from their first postdoc before possibly doing another one. Reply: Currently we reported on the total number of publications. We now improve the labeling and add a column for number publications during the postdoc. Unfortunately, we do not have the data about publications per postdoc fellowship. Minor concerns: “Their research requires heavier funding” - could the authors articulate what they mean here? Is it that research generally has become more expensive? Or is there some increased burden at the postdoctoral position? Reply: This refers to the burden of maintaining a lab and conducting top-quality scientific work. We are now more explicit in the text and add a supporting citation. The increasing cost of research and the market forces that are resulting in more postdocs (that they are essentially cheaper than graduate students) have been pointed out by Paula Stephan in her book “How Economics Shapes Science”7 and elsewhere, and these works should be cited appropriately in the introduction Reply: Thank you for this valuable source. We now included it in the introduction. “Moreover, postdocs that are not able to achieve an academic appointment often become over-qualified for industry positions while losing alternative higher compensation (salaries in the industry)” - Kahn and Ginther 20171should be cited here. Reply: Thank you! Footnote 3 should probably be moved into the Figure Legend of Figure 1, to highlight transformation of the 1-7 scale into a 1-10 scale. Reply: Given reviewers’ feedback we have decided to remove Figure 1. In Table 1, “Duration of postdoc (years)” may be better described as “Total years in postdoctoral positions” or similar, if the survey questions are understood correctly - it is not clear whether this is pointing out the years in the current postdoc, or the years of postdoccing in total. Reply: Done. Supplementary File 2 is not as described, but is the same as Supplementary File 1. Reply: We now corrected this. Thank you. References 1. Kahn S, Ginther DK: The impact of postdoctoral training on early careers in biomedicine.Nat Biotechnol. 2017; 35 (1): 90-94 PubMed Abstract \| Publisher Full Text 2. Sauermann H, Roach M: SCIENTIFIC WORKFORCE. Why pursue the postdoc path?. Science. 2016; 352 (6286): 663-4 PubMed Abstract \| Publisher Full Text 3. Heggeness ML: Preparing for the 21st Century Biomedical Research Job Market: Using Census Data to Inform Policy and Career Decision-Making. Self-Journals of Science. 2016. Publisher Full Text 4. Mason JL, Johnston E, Berndt S, Segal K, Lei M, Wiest JS: Labor and skills gap analysis of the biomedical research workforce.FASEB J. 2016; 30 (8): 2673-83 PubMed Abstract \| Publisher Full Text 5. Faupel-Badger JM, Nelson DE, Izmirlian G: Career Satisfaction and Perceived Salary Competitiveness among Individuals Who Completed Postdoctoral Research Training in Cancer Prevention.PLoS One. 2017; 12 (1): e0169859 PubMed Abstract\| Publisher Full Text 6. Roach M, Sauermann H: The declining interest in an academic career.PLoS One. 2017; 12 (9): e0184130 PubMed Abstract \| Publisher Full Text 7. Stephan P: How Economics Shapes Science. Harvard University Press, Cambridge, MA, USA. 2012." } ] }, { "id": "26675", "date": "16 Oct 2017", "name": "Jennifer M. Miller", "expertise": [ "Reviewer Expertise Science and technology policy", "public 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\nThis review will focus a critical eye on three issues, integration with existing literature, survey methodology, and use of the Satisfaction with Life Scale (SWLS). The review then acknowledges some points of agreement and support for the authors and ultimately recommends significant revision for publication, with some reservations.\nThe abstract begins with the implication that the public discourse about postdocs is not informed by empirical work. The implication that empirical work is missing is a red flag to me whenever I encounter it, not only when it is my own empirical work that is being overlooked. Yet I do believe that our paper (Miller & Feldman, 2015) that uses the Science and Engineering PhD and Postdoc Survey (SEPPS) conducted by Sauermann and Roach to study dissatisfaction with postdoc appointments could have informed the design and interpretation of this study. So at the risk of being a caricature of reviewers who plug their own work, I will begin by pointing the authors toward this paper, which also contains a summary of prior research on postdoc satisfaction. The Scaffidi and Berman (2011) paper is also very relevant to the topic.\nI was pleased to see that the authors had cited Davis’s 2009 chapter analyzing the Sigma Xi survey of postdocs, as this has been some of the most influential empirical work on the postdoc experience. As a methodological step to avoid this type of disconnect from prior literature, I would recommend the practice of using Google Scholar’s “cited by” feature to look at the studies that have cited this chapter. It will connect the authors to even more recent and relevant empirical work.\nFurther review of existing literature would also have been helpful to the authors in their use of the SWLS. Diener and colleagues have published a number of subsequent review articles (including but not limited to Diener & Pavot 1993 and Pavot & Diener 2008), but this study relies on the original 1985 paper.\nThe review will now turn to issues of interpretation related to the survey data and measurement scales.\nInterpretation of the survey data was complicated by the absence of a correlation table. It was not entirely clear, but it appears that conclusions are drawn from correlations, rather than regression analyses that more carefully model the relationship to satisfaction by including control variables. I recommend the authors clearly explain the analyses performed and provide a correlation table. I have two concerns regarding the representativeness of the sample. First, the authors say the survey was distributed by postdoc associations to their members. Postdoc associations do not necessarily include all postdocs at an institution and often serve as agents of change, sometimes including formal unionization efforts. This is a limitation that would discourage me from drawing policy conclusions from the study. Postdoc association membership lists may overrepresent dissatisfied postdocs. Of course, it is also possible that at least some postdoc associations have a broader mailing list. It is even possible that not all postdoc associations distributed the survey at all. With only 29 associations, it seems likely it would be feasible to contact them to confirm distribution and to give some consideration to their mission statements.\nSecond, in analyzing survey data, it is a questionable choice to include only complete cases. Various techniques for imputation allow the researchers to avoid discarding the valuable data from incomplete cases. The fact that age and gender were missing from over 20% of cases, while other variables were provided more completely, suggests that there may be some bias introduced by discarding missing data. Response bias also tends to overrepresent those with strong feelings and opinions.\nThe likelihood for sample bias due to the reliance on postdoc associations is my main concern with the study as a whole. This cannot be remedied but can be acknowledged as an important limitation.\nComparison of postdocs’ SWLS scores with early studies of relevant populations was complicated by the fact that the authors report the mean (4.47 on a 1-7 scale), while apparently other studies tend to report the total scale score (5-35). On page 2, the authors refer to 4 as the median, when 4 is more accurately described as the midpoint of the scale. Diener & Pavot (1993) are careful to interpret findings relevant to their scale anchors. A mean of 4.47 indicates a neutral to slightly satisfied group. But taking into account confidence intervals, it is not clear to me that the satisfaction of postdocs is significantly worse than that found by Diener & Pavot in 1993 for several groups of US college students. Given the cultural variation in reporting life satisfaction, the absence of nationality data also complicates interpretation of the scale (note the low satisfaction for Chinese students reported in the 1993 paper). Postdocs are a highly international group and nationality has been found to be relevant to satisfaction (Sabharwal & Corley 2009).\nFigure 1 seemed especially problematic in terms of scale interpretation. This measurement seems to be on a different scale entirely, as it has a category >7, which would be impossible on a 1-7 scale. The authors should review literature about and applications of the SWLS to relevant populations more thoroughly and contextualize their findings in clear, comparable terms.\nOverall I did not find support for the claim that this survey showed surprisingly low levels of wellbeing.\nI’d like to conclude with a few points of agreement with prior research. For example, we also found that satisfaction does typically decline the longer someone is a postdoc (Miller & Feldman 2015). It is also not surprising that people lost interest in pursuing tenure-track academic appointments. If they did not lose interest, the competition for the relatively few available positions would be even more fierce, with perhaps even more stark disappointment among those not selected. Yet, while this competition has intensified, it is by no means new. Like entertainment and athletics, scientific careers function as tournaments (Freeman et al. 2001). I agree with the authors’ recommendation that doctoral students and postdocs would benefit from more awareness of the structure of the scientific labor market.\nPrior research also supports the focus on improving the postdoc experience through interpersonal, lab-level interventions, rather than more tangible issues of pay and benefits (Miller & Feldman 2015; Scaffidi & Berman, 2011; Davis 2009). Research on postdoc satisfaction tends to identify the mentoring relationship and professional development as key to postdocs’ satisfaction.\nIn summary, I have some fairly strong methodological reservations about this paper. As presented, the findings of dissatisfaction do not seem particularly strong. There are reasons to question the role of sample bias in selecting for more dissatisfied postdocs. However, I could support publication of a revised version that better contextualizes the paper with existing research, acknowledges the activist role of postdoc associations and resulting potential for bias, better examines the potential for nonresponse bias and missing data, and provides careful comparisons with other studies using the SWLS.\n\nMinor points\n\nFor policy-relevant research, it is useful to report the time frame during which the data were collected. Supplementary File 1 is described as having been distributed among 190 postdocs. In fact, it was completed by 190 postdocs and it is unknown to how many the survey was distributed. The NSF Survey of Graduate Students and Postdoctorates in Science and Engineering could provide an estimate of the number of postdocs at these institutions. I do not usually use SPSS, so was not able to examine the data file. For what is probably a fairly manageable dataset like this, it would be helpful to provide a flat text file in a format like .csv. Both supplementary files appear to link to the survey, not the list of postdoc associations. Paragraph 1, “warranted” seems like an odd word choice here.\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? No", "responses": [ { "c_id": "3613", "date": "02 May 2018", "name": "Amir Grinstein", "role": "Author Response", "response": "Referee 1 Jennifer M. Miller, USC Sol Price School of Public Policy, University of Southern California, Los Angeles, CA, USA Approved with Reservations This review will focus a critical eye on three issues, integration with existing literature, survey methodology, and use of the Satisfaction with Life Scale (SWLS). The review then acknowledges some points of agreement and support for the authors and ultimately recommends significant revision for publication, with some reservations. The abstract begins with the implication that the public discourse about postdocs is not informed by empirical work. The implication that empirical work is missing is a red flag to me whenever I encounter it, not only when it is my own empirical work that is being overlooked. Yet I do believe that our paper (Miller & Feldman, 2015) that uses the Science and Engineering PhD and Postdoc Survey (SEPPS) conducted by Sauermann and Roach to study dissatisfaction with postdoc appointments could have informed the design and interpretation of this study. So at the risk of being a caricature of reviewers who plug their own work, I will begin by pointing the authors toward this paper, which also contains a summary of prior research on postdoc satisfaction. The Scaffidi and Berman (2011) paper is also very relevant to the topic. I was pleased to see that the authors had cited Davis’s 2009 chapter analyzing the Sigma Xi survey of postdocs, as this has been some of the most influential empirical work on the postdoc experience. As a methodological step to avoid this type of disconnect from prior literature, I would recommend the practice of using Google Scholar’s “cited by” feature to look at the studies that have cited this chapter. It will connect the authors to even more recent and relevant empirical work. Further review of existing literature would also have been helpful to the authors in their use of the SWLS. Diener and colleagues have published a number of subsequent review articles (including but not limited to Diener & Pavot 1993 and Pavot & Diener 2008), but this study relies on the original 1985 paper. Reply: Thank you very much for directing us to highly relevant literature that we have overlooked. This is indeed an emerging and evolving area and we have now extended our literature search based on your guidance as well as integrated the above work into the revised manuscript. The review will now turn to issues of interpretation related to the survey data and measurement scales. Interpretation of the survey data was complicated by the absence of a correlation table. It was not entirely clear, but it appears that conclusions are drawn from correlations, rather than regression analyses that more carefully model the relationship to satisfaction by including control variables. I recommend the authors clearly explain the analyses performed and provide a correlation table. Reply: We now include a correlation matrix. We also ran regression analysis on which we report in the revised version. I have two concerns regarding the representativeness of the sample. First, the authors say the survey was distributed by postdoc associations to their members. Postdoc associations do not necessarily include all postdocs at an institution and often serve as agents of change, sometimes including formal unionization efforts. This is a limitation that would discourage me from drawing policy conclusions from the study. Postdoc association membership lists may overrepresent dissatisfied postdocs. Of course, it is also possible that at least some postdoc associations have a broader mailing list. It is even possible that not all postdoc associations distributed the survey at all. With only 29 associations, it seems likely it would be feasible to contact them to confirm distribution and to give some consideration to their mission statements. Second, in analyzing survey data, it is a questionable choice to include only complete cases. Various techniques for imputation allow the researchers to avoid discarding the valuable data from incomplete cases. The fact that age and gender were missing from over 20% of cases, while other variables were provided more completely, suggests that there may be some bias introduced by discarding missing data. Response bias also tends to overrepresent those with strong feelings and opinions. The likelihood for sample bias due to the reliance on postdoc associations is my main concern with the study as a whole. This cannot be remedied but can be acknowledged as an important limitation. Reply: We have decided to focus on postdoc associations given they represent many postdocs and enabled us one access point to large number of potential survey participants. Following your concern we now acknowledge this potential limitation in our GD. About the missing data of gender and age: first, it seems reasonable to assume that missing data is equally distributed across the different postdoc associations and disciplines. Further, as these variables are only control variables rather than at the heart of our conceptualization we hope this limitation is not critical. Finally, it is important for us to clarify that we did use all the data available (we didn’t “throw” participant if s/he missed one of the questions). Comparison of postdocs’ SWLS scores with early studies of relevant populations was complicated by the fact that the authors report the mean (4.47 on a 1-7 scale), while apparently other studies tend to report the total scale score (5-35). On page 2, the authors refer to 4 as the median, when 4 is more accurately described as the midpoint of the scale. Diener & Pavot (1993) are careful to interpret findings relevant to their scale anchors. A mean of 4.47 indicates a neutral to slightly satisfied group. But taking into account confidence intervals, it is not clear to me that the satisfaction of postdocs is significantly worse than that found by Diener & Pavot in 1993 for several groups of US college students. Given the cultural variation in reporting life satisfaction, the absence of nationality data also complicates interpretation of the scale (note the low satisfaction for Chinese students reported in the 1993 paper). Postdocs are a highly international group and nationality has been found to be relevant to satisfaction (Sabharwal & Corley 2009). Figure 1 seemed especially problematic in terms of scale interpretation. This measurement seems to be on a different scale entirely, as it has a category >7, which would be impossible on a 1-7 scale. The authors should review literature about and applications of the SWLS to relevant populations more thoroughly and contextualize their findings in clear, comparable terms. Overall I did not find support for the claim that this survey showed surprisingly low levels of wellbeing. Reply: This is a valuable discussion to have. Thank you. First, we used the 1-7 scale to be able to compare our results to some of the prior studies that did report their results on a 1-7 scale (e.g., Kjeldstadli et al., 2006; Samaranayake and Fernando, 2011). While locating the 4.47 mean on the 5-35 SWLS scale suggests that our score falls under the “slightly satisfied” category (score = 22.35; Diener and Pavot, 1993) the finding is the lowest compared to all other student groups in developed countries or professionals (apart from “elderly caregivers”) as can be seen from Table 1 in Diener and Pavot (1993). According to these authors, satisfaction with life in the developed world is likely to hit “slightly satisfied” as a starting point. In this context our results actually show a surprisingly low satisfaction with life of postdocs. We now integrate this discussion in the text. Your point about the diverse cultures and countries postdocs come from and how this plays a role is a very valid one and we are now discussing this as an avenue for future research. We now use the term “midpoint” rather than “median” when addressing the score 4. The decision to adapt our scale for the purpose of the figure appeared in footnote 3 in the original version of the manuscript: “To be compatible with Abdallah et al.’s (2009) map, we transformed our 1-7 satisfaction with life scale to a 1-10 scale, on which the surveyed postdocs demonstrate a 6.1 satisfaction with life score.” Still, following the feedback from the review team we have decided to remove Figure 1 from the revised version. I’d like to conclude with a few points of agreement with prior research. For example, we also found that satisfaction does typically decline the longer someone is a postdoc (Miller & Feldman 2015). It is also not surprising that people lost interest in pursuing tenure-track academic appointments. If they did not lose interest, the competition for the relatively few available positions would be even more fierce, with perhaps even more stark disappointment among those not selected. Yet, while this competition has intensified, it is by no means new. Like entertainment and athletics, scientific careers function as tournaments (Freeman et al. 2001). I agree with the authors’ recommendation that doctoral students and postdocs would benefit from more awareness of the structure of the scientific labor market. Prior research also supports the focus on improving the postdoc experience through interpersonal, lab-level interventions, rather than more tangible issues of pay and benefits (Miller & Feldman 2015; Scaffidi & Berman, 2011; Davis 2009). Research on postdoc satisfaction tends to identify the mentoring relationship and professional development as key to postdocs’ satisfaction. Reply: These are important points and highly relevant sources. We connect them now with our findings and implications. In summary, I have some fairly strong methodological reservations about this paper. As presented, the findings of dissatisfaction do not seem particularly strong. There are reasons to question the role of sample bias in selecting for more dissatisfied postdocs. However, I could support publication of a revised version that better contextualizes the paper with existing research, acknowledges the activist role of postdoc associations and resulting potential for bias, better examines the potential for nonresponse bias and missing data, and provides careful comparisons with other studies using the SWLS. Reply: Thank you. We have made an attempt to address all your comments and benefit significantly from your valuable feedback. Minor points For policy-relevant research, it is useful to report the time frame during which the data were collected. Reply: We now add the time frame of the study. Supplementary File 1 is described as having been distributed among 190 postdocs. In fact, it was completed by 190 postdocs and it is unknown to how many the survey was distributed. The NSF Survey of Graduate Students and Postdoctorates in Science and Engineering could provide an estimate of the number of postdocs at these institutions. Reply: First, we change the wording to “completed by”. Also, we now refer to the overall sample frame of postdocs in footnote 1. I do not usually use SPSS, so was not able to examine the data file. For what is probably a fairly manageable dataset like this, it would be helpful to provide a flat text file in a format like .csv. Reply: We have now submitted both SPSS and Excel files. Both supplementary files appear to link to the survey, not the list of postdoc associations. Reply: We made sure this is corrected. Paragraph 1, “warranted” seems like an odd word choice here. Reply: We have modified the sentence. References 1. Davis.G: Improving the postdoctoral experience: An empirical approach. In Science and engineering careers in the United States: An analysis of markets and employment. University of Chicago Press. 2009. 99-127 Reference Source 2. Freeman R, Weinstein E, Marincola E, Rosenbaum J, Solomon F: Competition and careers in biosciences. Science. 2001; 294 (5550): 2293-2294 Publisher Full Text 3. Miller J.M, Feldman M.P: isolated in the Lab: examining Dissatisfaction with Postdoctoral Appointments. The Journal of Higher Education. 2015; 86 (5): 697-724 Reference Source 4. Sabharwal M: Faculty job satisfaction across gender and discipline. The Social Science Journal. 2009; 46 (3): 539-556 Reference Source 5. Scaffidi A.K: A positive postdoctoral experience is related to quality supervision and career mentoring, collaborations, networking and a nurturing research environment. Higher Education. 2011; 62 (6): 685 Reference Source 6. Pavot W: Review of the satisfaction with life scale. Psychological assessment. 1993; 5 (2): 164 Reference Source 7. Pavot W: The satisfaction with life scale and the emerging construct of life satisfaction. The Journal of Positive Psychology. 2008; 3 (2): 137-152 Reference Source" } ] } ]	1	https://f1000research.com/articles/6-1642
https://f1000research.com/articles/7-520/v1	30 Apr 18	{ "type": "Case Report", "title": "Case Report: Kikuchi: The great mimicker", "authors": [ "Kevin Bryan Lo", "Anna Papazoglou", "Lorayne Chua", "Nellowe Candelario", "Anna Papazoglou", "Lorayne Chua", "Nellowe Candelario" ], "abstract": "Kikuchi-Fujimoto disease is a form of a benign necrotizing lymphadenitis which is most commonly misdiagnosed as tuberculosis and or lymphoma, usually more common among young adults in Asia. It is a benign disease but can mimic a lot of other disease processes spanning infectious, rheumatologic and even hematologic malignancies. Our patient presented with prolonged fever and lymphadenopathy. Initial considerations were lymphoma and a nonspecific viral infection. A CT scan showed diffuse cervical lymphadenopathy with lacrimal gland involvement. An excisional lymph node biopsy was done which revealed Kikuchi disease. Patient was given steroids with immediate response with defervescence. Kikuchi is a disease with many mimics and a complete workup is needed to exclude serious disease like malignancy.", "keywords": [ "lymphadenitis", "fever", "Kikuchi", "autoimmune" ], "content": "Introduction\n\nKikuchi-Fujimoto disease (sometimes also known as Kikuchi Disease) is usually more common among young adults in Asia. It is a benign disease, but can mimic a lot of other disease processes spanning infectious, rheumatologic and even hematologic malignancies1. It usually presents with fever and cervical lymphadenopathy but occasionally it can manifest together with other unusual symptoms further increasing the chances of misdiagnosis.\n\n\nCase\n\nA 20-year-old African American woman with no other known prior medical history, presented to our institution January 2018 with fevers of 3 weeks’ duration. The fevers were predominantly in the late afternoon hours, associated with night sweats, frontal headache, tender cervical lymphadenopathy, anorexia and malaise.\n\nTwo weeks prior she saw her primary care physician who diagnosed her with viral illness and recommended supportive care. She also visited the emergency department and was diagnosed with lymphadenitis; a course of amoxicillin/clavulanic acid was prescribed of unrecalled dose and she wasn’t able to finish the whole course. Symptoms however persisted, and the patient also developed bilateral periorbital swelling and non-bloody diarrhea prompting her presentation at our institution. The patient indicated they had no cough, chest pain, dysuria, abdominal pain, arthralgia, rash, recent travel or sick contacts.\n\nThe patient was not in distress, with blood pressure of 120/70 mm Hg, febrile to 39.6 C and tachycardic with heart rate of 110 bpm. Physical exam was notable for bilateral periorbital swelling with violet discoloration of the eyelids, conjunctival pallor and painless bilateral cervical lymphadenopathy. No rash or joint swelling was noted.\n\nComplete blood count revealed leukopenia with a white cell count of 2.9 × 103 /mcL (65% neutrophils, 13% lymphocytes, 13% bands), microcytic anemia with a hemoglobin of 8.5 gr/dL (mean corpuscular volume 65 fL) and 181 × 103 /mcL platelets. C-reactive protein (CPR) and erythrocyte sedimentation rate (ESR) level were markedly elevated at 51 and 84 respectively. Lactate dehydrogenase (LDH), ferritin and haptoglobin were also elevated. The patient tested negative for β-human chorionic gonadotropin (hCG), HIV, hepatitis B and C, angiotensin converting enzyme (ACE), antinuclear antibodies (ANA) and rheumatoid factor (RF).\n\nComputed Tomography of the neck revealed bilateral cervical lymphadenopathy, enhancement and mild enlargement of the parotid and lacrimal glands and diffuse swelling of the pharyngeal mucosa and marked enhancement of bilateral cervical soft tissue planes [Figure 1 and Figure 2].\n\nBilateral lacrimal glands appear large with mild increased enhancement.\n\nBilateral left greater than right cervical jugular chain, level I, occipital and supraclavicular lymph nodes demonstrate heterogeneous enhancement and enlargement, largest demonstrating conglomeration and cystic changes along the left jugular chain measuring up to 8.5 cm in length of the conglomerate.\n\nShe was observed off antibiotics. Blood cultures, serology for Epstein Barr virus (EBV) and cytomegalovirus (CMV), bone marrow biopsy and flow cytometry were all negative. Excisional biopsy of the left cervical lymph node revealed characteristic findings of Kikuchi-Fujimoto disease which showed geographic necrosis with fibrinoid deposits and apoptotic cells surrounded by a mononuclear infiltrate characteristically without neutrophils and eosinophils [Figure 3]. The patient was started on prednisone 40mg per day with rapid resolution of symptoms. Steroids were tapered after one week of treatment. Upon follow up in Rheumatology clinic 4 months later, patient was noted to be completely symptom free.\n\nGeographical necrosis with fibrinoid deposits and nuclear fragments with apoptotic cells. Surrounding this area are pale histiocytes and lymphocytes. Neutrophils and eosinophils are characteristically absent.\n\n\nDiscussion\n\nKikuchi disease was first independently described through case series in 1972 by Kikuchi and Fujimoto as a form of a benign necrotizing lymphadenitis which was most commonly misdiagnosed as tuberculosis and or lymphoma2. The main etiology for Kikuchi disease is still unknown but there are various studies that implicate viruses such as EBV as a potential trigger2,3. It is also closely related to systemic lupus erythematosus (SLE) and in fact, there are studies and case reports showing a strong association between the two disease processes with the diagnosis of SLE coming before, after or even simultaneously with Kikuchi disease4,5. The most frequent presenting symptom was fever while the most common presenting sign was cervical lymphadenopathy5. It also presents together with constitutional symptoms like night sweats and weight loss which can be initially be misdiagnosed as tuberculosis or lymphoma1,2. However, Kikuchi disease has also been implicated to cause a wide range of symptoms ranging from neurological, musculoskeletal, cutaneous and glandular dysfunction6. Eye manifestations for Kikuchi usually present as uveitis and conjunctivitis6,7. Our case is unique because bilateral eyelid swelling has only been reported twice in the literature as a possible presentation of Kikuchi disease, this may be attributed to lacrimal gland involvement which was seen in the imaging findings in our patient6,8,9. Definitive diagnosis is established by lymph node biopsy. Classic biopsy findings include necrosis without a neutrophilic infiltrate with the predominance of histiocytes and T lymphocytes1,2. Kikuchi is a benign self-limiting disease and symptoms usually resolves spontaneously within 4 months in majority of cases with supportive treatment1,6. The use of glucocorticoids have been found to have some benefit but is usually reserved in more severe persistent cases5,6,10. Kikuchi is a disease with a lot of mimics, the amount of workup alone together with the actual disease manifestations can lead to a lot of morbidity and discomfort for the patient. Nevertheless, a complete workup including an excisional biopsy is recommended to help rule out other serious diseases like malignancy. Close follow up is also needed to monitor for the development of closely associated rheumatological diseases like SLE. Strengths in the approach of the case was the exhaustive diagnostic approach used to arrive at the correct diagnosis for the patient. All possible differentials were considered especially the serious ones such as malignancy. Weakness involved were due to the extensive workup done which consisted of numerous blood tests and invasive tests such as a biopsy, this caused a significant degree of anxiety and morbidity to the patient as well.\n\n\nConclusions\n\nKikuchi is a great mimicker and can be confused with tuberculosis, lymphoma and other viral illnesses. A complete workup including an excisional biopsy is recommended to help rule out other serious diseases like malignancy. Close follow up is needed to monitor for the development of closely associated rheumatological diseases like SLE.\n\n\nEthics and consent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient and parent.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "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\nHutchinson CB, Wang E: Kikuchi-Fujimoto disease. Arch Pathol Lab Med. 2010; 134(2): 289–93. PubMed Abstract\n\nShirakusa T, Eimoto T, Kikuchi M: Histiocytic necrotizing lymphadenitis. Postgrad Med J. 1988; 64(748): 107–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChiu CF, Chow KC, Lin TY, et al.: Virus infection in patients with histiocytic necrotizing lymphadenitis in Taiwan. Detection of Epstein-Barr virus, type I human T-cell lymphotropic virus, and parvovirus B19. Am J Clin Pathol. 2000; 113(6): 774–81. PubMed Abstract \| Publisher Full Text\n\nBaenas DF, Diehl FA, Haye Salinas MJ, et al.: Kikuchi-Fujimoto disease and systemic lupus erythematosus. Int Med Case Rep J. 2016: 9: 163–167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKucukardali Y, Solmazgul E, Kunter E, et al.: Kikuchi-Fujimoto Disease: analysis of 244 cases. Clin Rheumatol. 2007; 26(1): 50–4. PubMed Abstract \| Publisher Full Text\n\nBosch X, Guilabert A, Miquel R, et al.: Enigmatic Kikuchi-Fujimoto disease: a comprehensive review. Am J Clin Pathol. 2004; 122(1): 141–52. PubMed Abstract \| Publisher Full Text\n\nGalor A, Georgy M, Leder HA, et al.: Papillary conjunctivitis associated with Kikuchi disease. Cornea. 2008; 27(8): 944–6. PubMed Abstract \| Publisher Full Text\n\nGuerriero S, Ruggeri E, Piscitelli D, et al.: Kikuchi-Fujimoto disease: a rare case of orbital involvement. Orbit. 2012; 31(6): 420–2. PubMed Abstract \| Publisher Full Text\n\nChavis PS, Fallata A, Al-Hussein H, et al.: Lacrimal gland involvement in Kikuchi-Fujimoto disease. Orbit. 1998; 17(2): 113–7. PubMed Abstract \| Publisher Full Text\n\nJang YJ, Park KH, Seok HJ: Management of Kikuchi’s disease using glucocorticoid. J Laryngol Otol. 2000; 114(9): 709–11. PubMed Abstract \| Publisher Full Text" }	[ { "id": "35130", "date": "25 Jun 2018", "name": "Abdou-Rajack Ndiaye", "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 is correctly written. It is an original one. The described pathology is uncommon and raises diagnostic problems. References are up-to-date.\nHowever, some minor revisions must be done:\nSerodiagnostic tests for toxoplasmosis, rubella, syphlis, and infectious mononucleosis must be performed in presence of chronic lymphadenopathy with fever, even they are often negative in such case.\n\nPrecise the place of immunohistochemical study (cd68, cd8) to confirm the diagnosis.\n\nDeaths are rare but have been observed in systemic forms of kikuchi disease.\n\nRecent treatment with intravenous immunoglobulins has been proposed in severe and resistant forms.\n\nFigure 3: Specify the used coloration and the corresponding magnification.\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": "35937", "date": "07 Aug 2018", "name": "Arvind P. Ganpule", "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\nExplanation for second referee question:\n\nPeriodic acid-Schiff (PAS) stain , Ziehl Neelsen (ZN) stain, Genexpert examination for tubercle bacilli (TB), immmunohistochemistry (IHC) examination for lymphoma, tests for systemic lupus erythematosus (SLE) to rule out different differentials, were not mentioned.\n\nKey features on lymph node biopsy are fragmentation, necrosis and karyorrhexis were not mentioned by authors in the histopathology examination.\nTreatment includes symptomatic care, analgesics-antipyretics, corticosteroids and not the corticosteroid alone as mentioned by authors.\nAuthor may put “Ganpule AP, Chabra JS, Singh AG, Tak GR, Soni S, Sabnis R, Desai M. Case Report: Kikuchi-Fujimoto disease: a diagnostic and therapeutic dilemma following pretransplant nephrectomy for a 2.35 Kg kidney. F1000Research. 2016;5” as the reference for their article.\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? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-520
https://f1000research.com/articles/7-518/v1	30 Apr 18	{ "type": "Case Report", "title": "Case Report: My lung broke my heart! Takotsubo cardiomyopathy due to pneumonia", "authors": [ "Navid Ahmed", "Himali Gandhi", "Daniel B. Sims", "Himali Gandhi", "Daniel B. Sims" ], "abstract": "Takotsubo cardiomyopathy (TTC), also known as stress-induced cardiomyopathy, is a cardiac syndrome that often mimics acute myocardial infarction. TTC is commonly triggered by physical or emotional stress; however, acute infection is a rarer etiology. This report concerns the case of an 82-year-old female who presented with non-positional and non-pleuritic chest pain, with an associated fever and cough and chest x-ray findings consistent with pneumonia. Cardiac enzymes and ECG findings were consistent with acute coronary syndrome (ACS); however, during coronary angiography, no coronary artery disease could explain the patient’s ACS. A post-catheterization echocardiogram revealed an ejection fraction of 25%, with apical akinesis. A repeat echocardiogram 4 weeks after presentation showed a normal EF and normal wall motion, confirming a diagnosis of TTC.", "keywords": [ "Takotsubo cardiomyopathy", "Heart failure", "Sepsis", "Geriatrics", "Cardiology", "Pneumonia" ], "content": "Introduction\n\nTakotsubo cardiomyopathy (TTC) is an etiology of chest pain that often mimics acute myocardial infarction. However, TTC presents with transient systolic dysfunction, which normalizes over time. Patients who typically present with TTC have an inciting physical or emotional stress event that is pinpointed as the etiology2.\n\nTTC is typically not associated with an infectious etiology as the inciting stressor; however, it has been rarely reported in previous case reports1. Acute infection should be increasingly recognized as a possible trigger of TTC in a patient with chest pain.\n\n\nCase presentation\n\nAn 82-year-old female from a nursing home, with a history of dementia, hypertension, hyperlipidemia and coronary artery disease (CAD), and a drug-eluting stent placed in the left circumflex artery, presented from nursing home with recurrent non-positional and non-pleuritic chest pain, along with associated fever and cough. Further medical history could not be obtained from the patient owing to underlying dementia. Of note, the patient had a normal echocardiogram 10 days prior to presentation, with an ejection fraction (EF) of 60%. On presentation, her blood pressure was 113/78 mm Hg, and she had a pulse of 137 beats/min and a temperature of 101°F (38.3°C).\n\nAn electrocardiogram (ECG) showed ST elevations in I, avL, and V3–V6 (Figure 1). A chest x-ray revealed pneumonia in the right middle and right lower lobes. The patient’s initial creatinine phosphokinase and troponin T were elevated at 330 U/l and 0.86 ng/ml, respectively, and ultimately peaked 6 h after presentation at 470 U/l and 1.39 ng/mll, respectively (normal creatinine phosphokinase < 200 U/l, normal Troponin T < 0.10 ng/ml.)\n\nThe patient was taken for an emergency cardiac catheterization after verification of goals of care. Left ventriculogram showed a hypercontractile base and apical akinesis, with an EF of 20% (Figure 2). There was no CAD, which explained the patient’s ECG or wall motion abnormalities, so no revascularization was performed.\n\nThe patient was placed on vancomycin (1 g every 12 h) and piperacillin–tazobactam (0.375 g every 6 h) for the treatment of pneumonia for a 7-day course, with an improvement in symptoms observed. She was continued on daily 81 mg aspirin, 50 mg metoprolol and 40 mg simvastatin, and started on 5 mg lisinopril. A repeat echocardiogram 4 weeks later revealed a normal EF and normal wall motion, confirming a diagnosis of TTC.\n\n\nDiscussion\n\nTTC, also known as stress-induced cardiomyopathy, is a cardiac syndrome that often mimics acute myocardial infarction and presents with transient systolic dysfunction of the apical segment of the left ventricle (LV), without the presence of obstructive coronary artery disease1. The syndrome has a higher incidence in women than men1.\n\nPatients typically present with chest pain and dyspnea; however, cases have been reported of syncope, palpitations, hypotension and shock as the initial manifestation of TTC. Typically, TTC is preceded by a stressful event, such as tragic personal news, assaults, arguments or accidents2. However, acute infection has been described as an uncommon etiology1,3–5. Typical ECG findings include ST segment elevations and T-wave inversions. Coronary angiography typically does not reveal a culprit lesion and LV angiography shows LV apical ballooning.\n\nThe pathophysiological basis of TTC is still unknown, although potential mechanisms include multi-vessel coronary vasospasm, coronary microvascular dysfunction and catecholamine cardiotoxicity1,2,6. TTC is commonly triggered by physical or emotional stress; however, rare cases of stress cardiomyopathy from acute infection have been reported1–3. A systematic review of sepsis and TTC hypothesized that inflammatory markers, such as tumor necrosis factor-α and interleukin-1β, along with other cytokines, act as a trigger for cardiac sympathetic nerve discharge, leading to an elevated norepinephrine state and then myocardial dysfunction2. Another possible mechanism of TTC is myocardial ischemia due to inadequate coronary blood flow during sepsis3. The patient in the present report presented with a baseline altered mental status secondary to dementia; no trigger for her episode of TTC other than her infection could be found.\n\nTTC is a reversible cardiomyopathy that is typically associated with emotional stress; however, other inciting factors can trigger TTC. Infection is an uncommon inciting event for TTC. In patients who present with signs and symptoms of acute infection and chest pain, TTC should be considered in the differential diagnosis during the evaluation and workup of the patient.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and images was obtained from a relative of the patient owing to the underlying dementia of 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\nDing H, Huang R, Shi X, et al.: Stress-induced cardiomyopathy following infection of the upper respiratory tract in an elderly female patient: A case report. Exp Ther Med. 2016; 12(5): 3083–3086. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPilgrim TM, Wyss TR: Takotsubo cardiomyopathy or transient left ventricular apical ballooning syndrome: A systematic review. Int J Cardiol. 2008; 124(3): 283–292. PubMed Abstract \| Publisher Full Text\n\nY-Hassan S, Settergren M, Henareh L: Sepsis-induced myocardial depression and takotsubo syndrome. Acute Card Care. 2014; [cited April 1, 2017]; 16(3): 102–109. PubMed Abstract \| Publisher Full Text\n\nClemente G, Tuttolomondo A, Colomba D, et al.: When sepsis affects the heart: A case report and literature review. World J Clin Cases. 2015; 3(8): 743–750. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrishnagopalan S, Kumar A, Parrillo JE, et al.: Myocardial dysfunction in the patient with sepsis. Current Opinion In Critical Care. 2002; 8(5): 376–388. PubMed Abstract \| Publisher Full Text\n\nCappelletti S, Ciallella C, Aromatario M, et al.: Takotsubo Cardiomyopathy and Sepsis. Angiology. 2017; 68(4): 288–303. PubMed Abstract \| Publisher Full Text" }	[ { "id": "58312", "date": "27 Jan 2020", "name": "Kunal Bhatt", "expertise": [ "Reviewer Expertise Heart failure", "transplant cardiology", "general cardiology", "TTE", "Amyloid cardiomyopathy" ], "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\nAhmed et al. report a concise case of an 82 year old female who developed Takotsubo cardiomyopathy (TTC) after an acute infectious bacterial Pneumonia.\nWhile TTC is usually described in relation to an emotional or physical stressor, this case is unique and contributes to the small existing literature of an infectious precipitant to the syndrome.\nThe clear objective data of an existing echo 10 days prior to admission showing a normal LVEF adds to the clear development of this patient's syndrome as being induced by her multi-lobar pneumonia.\nThis case report is written well with a clear focus on the objective of describing a unique case of TTC precipitated by multi-lobar Pneumonia. As sepsis and Pneumonia are fairly common diagnosis, this case report adds to the growing body of literature that describes infections as being another possible etiology of TTC in addition to emotinal and physical stressors.\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": "147688", "date": "06 Oct 2022", "name": "Nicola Marziliano", "expertise": [ "Reviewer Expertise Microbiology", "Cardiomiopathies", "Genetics", "Inherited cardiovascular disorders", "Channelopathies" ], "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\nTaken by itself, pneumonia does not affect directly the heart, but it is a lung infection caused by either bacteria (i.e. Klebsiella pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, etc ) or viruses (SARS-CoV-2, RSV, HxNx, etc) or even fungi. However, heart disease complications like congestive heart failure can cause a condition similar to pneumonia. Additionally, pneumonia tends to affect individuals who are also at high cardiovascular risk.\nAlthough acute cardiac events have been recognised as important complications in patients with pneumonia since the early 20th century, the magnitude of this problem has only recently begun to be appreciated fully. Under this light, the present work can add a further step in the disease (Takotsubo cardiomyopathy, TTC) understanding.\nAlthough, in the case presentation it is missing the link to the pneumonia symptoms and also the investigation of the underlying pneumonia’s causes (bacteria, viruses, fungi etc). I think the paper could be accepted after integrating shortly the clinical signs of the 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? 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": "150419", "date": "14 Oct 2022", "name": "Mohammad Ullah Firoze", "expertise": [ "Reviewer Expertise Clinical cardiology", "echocardiography", "Interventional cardiology." ], "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 and described case report.\nTCM in during severe infectious disease is not uncommon. This was reported some authors. Overall, this is a good case report to raise awareness about TCM during severe infectious diseases also.\nThere are two well-recognized diagnostic criteria for TCM. Mayo Clinic criteria and InterTAK criteria could have been discussed.\nEchocardiography before cardiac catheterization can give a good clue for diagnosis.\nThere are some ECG features, like lack of ST depression is suggestive of TCM in relation to ACS.\nMyocarditis and pheochromocytoma are two important differential diagnoses, this could be mentioned. Specially myocarditis should be excluded when there is an associated infectious disease like a viral disease. Overall, this is a good case report to raise awareness about TCM during severe infectious diseases also.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-518
https://f1000research.com/articles/7-280/v1	06 Mar 18	{ "type": "Opinion Article", "title": "The convergent epidemiology of tuberculosis and human cytomegalovirus infection", "authors": [ "Frank Cobelens", "Nico Nagelkerke", "Helen Fletcher", "Nico Nagelkerke", "Helen Fletcher" ], "abstract": "Although several factors are known to increase the risk of tuberculosis, the occurrence of tuberculosis disease in an infected individual is difficult to predict. We hypothesize that active human cytomegalovirus infection due to recent infection, reinfection or reactivation plays an epidemiologically relevant role in the aetiology of tuberculosis by precipitating the progression from latent tuberculosis infection to disease. The most compelling support for this hypothesis comes from the striking similarity in age-sex distribution between the two infections, important because the age-sex pattern of tuberculosis disease progression has not been convincingly explained. Cytomegalovirus infection and tuberculosis have other overlapping risk factors, including poor socio-economic status, sexual contact, whole blood transfusion and solid organ transplantation. Although each of these overlaps could be explained by shared underlying risk factors, none of the epidemiological observations refute the hypothesis. If this interaction would play an epidemiologically important role, important opportunities would arise for novel approaches to controlling tuberculosis.", "keywords": [ "Tuberculosis", "latent tuberculosis infection", "human cytomegalovirus", "epidemiology", "age pattern", "risk factor" ], "content": "Introduction\n\nWith 10.4 million new cases and 1.7 million deaths per year, tuberculosis (TB) remains a major global health problem1. Only 5%–15% of individuals infected with Mycobacterium tuberculosis (Mtb) ever develop TB disease, and over 50% of these do so within two years after infection2. Although risk factors for progression to TB disease have been identified3, disease occurrence cannot be accurately predicted4.\n\nRecent data suggest that infection with human cytomegalovirus (HCMV) is a predictor of TB disease in infants. In a cohort study of South African infants, an HCMV-specific IFN-γ T-cell response was associated with a 2.2-fold increased risk of TB disease over a period of up to 3 years. A similar response to Epstein-Barr virus (EBV) showed no such associations5. HCMV-positive and HCMV-negative infants had distinct immune pathways associated with TB disease. Although CD8+ T-cell activation was a distinguishing feature of HCMV-positive infants, the proposed immunological mechanism was impairment of the natural killer (NK) cell response. In African infants, HCMV infection induced profound CD8+ T-cell and NK cell differentiation and poor physical growth5–7.\n\nThe possibility that this association between HCMV and TB disease progression is causal, also holds in adults, and thus merits further study is dependent on its epidemiological plausibility. Only few published studies have investigated epidemiological associations between the two diseases8–10. Despite this paucity of direct evidence we argue that the epidemiology of TB and HCMV share important similarities that make HCMV infection a plausible candidate as a cause of TB disease progression.\n\n\nViral triggers of tuberculosis disease\n\nVarious etiological frameworks for TB disease progression have been developed. One proposed by Comstock considers TB disease the result of two hits or causes, one of which is Mtb infection, and the other (still) unknown11. In this framework, factors that strongly increase the risk of TB disease such as HIV infection or anti-tumour necrosis alpha therapy may act as a second hit but would not account for all or most TB cases.\n\nSeveral factors have been identified that increase the risk of disease progression, such as low body-mass index12, diabetes13, tobacco smoking14, and alcohol abuse15. As their effects are modest another framework has emerged that these are predisposing conditions for disease progression while other, yet unidentified precipitating events are needed to trigger progression to active disease16. Among the precipitating events suggested are respiratory viral infections, notably influenza A. Influenza A induces type I interferons that impair immunity against mycobacteria17. Also, notification of TB tends to peak in the months after winter when most respiratory viruses circulate18, and TB mortality has shown increases during influenza epidemics19,20. However, careful analysis of seasonality data suggests that it is TB transmission rather than disease progression that is increased in winter21. HCMV has been suggested in three studies from Nigeria, Russia and Uganda that all found higher prevalence or levels of IgG HCMV antibodies in diagnosed TB patients compared to healthy controls and patients diagnosed with other diseases8–10.\n\n\nHuman cytomegalovirus infection\n\nHCMV, human herpesvirus 5, is a double-stranded DNA virus. After primary infection, usually through mucosal contact, HCMV remains dormant in the host’s myeloid tissues but can reactivate if immunity is compromised. Primary infection is often asymptomatic but can present as mononucleosis with fever, pharyngo-tonsillitis and lymphadenopathy. In congenitally infected infants HCMV may cause severe generalized infection with high case fatality and neurologic sequelae22. Generalized infection also occurs in severely immunocompromised adults, usually through reactivation. During primary infection and reactivation virus is shed in the urine, saliva, breast milk, cervical fluid and semen23. Common routes of transmission are from mother to child during delivery, between children and by sexual contact. Transmission through blood transfusion and solid organ transplantations also occurs.\n\nHCMV viruses show genomic diversity, in particular in genes coding for envelope glycoproteins, and polymorphisms in these genes have been used to genotype strains24,25. Both immunocompromised and immunocompetent individuals can be re-infected and harbour multiple HCMV strains26–28.\n\nPrimary HCMV infection is characterized by profound expansion of antigen specific CD8+ and CD4+ T cells and NK cell populations with specificity for HCMV29. HCMV expanded NK cells can display inappropriate homing to tissue infected with other pathogens and lower IFN-γ secretion in response to pathogens30. HCMV has multiple immune evasion strategies29, which may make the microenvironment around latently infected myeloid cells suppressive to T-cell function, potentially creating an environment permissive for mycobacterial growth31.\n\nThe lung is a reservoir of HCMV infection and frequently the site of viral reactivation, which drives inflammation and in mice may cause pulmonary fibrosis32. It is therefore possible that replication of Mtb in the lungs could lead to reactivation of latent HCMV or that reactivation of latent HCMV could precipitate progression to TB disease.\n\n\nEpidemiological convergence\n\nBoth Mtb and HCMV infections are ubiquitous1,33, and during millions of years of co-evolution have become highly human host-specific34–36. An animal reservoir has been described for neither Mtb nor HCMV (several monkey and rodent species have their distinct CMV species), implying that their epidemiological patterns are entirely determined by transmission between, and carriage by, humans.\n\nWe hypothesize that immunologically active HCMV infection, whether primary, reactivation or re-infection, acts as (depending on one’s preferred framework) second-hit or precipitating factor for progression of latent TB infection to TB disease at an epidemiologically relevant scale. We base this on two arguments: their striking similarity in age distribution, and the existence of congruent risk factors [Box 1].\n\nWe systematically searched PubMed for the following combinations of keywords: tuberculosis and cytomegalovirus; cytomegalovirus and prevalence or seroprevalence; cytomegalovirus and age; cytomegalovirus and reinfection; cytomegalovirus and sexual; tuberculosis and sexual; tuberculosis and sexual transmitted infections or Chlamydia or gonorrhoea or human papillomavirus; tuberculosis and blood transfusion; cytomegalovirus and blood transfusion; tuberculosis and gastrectomy; cytomegalovirus and renal dialysis; tuberculosis and renal dialysis; tuberculosis and organ transplantation; cytomegalovirus and organ transplantation.\n\nWe in addition made use of an extensive review of the literature on age-sex distribution of tuberculosis incidence published by Nagelkerke (2012)37.\n\nThe probability of progressing from TB infection to disease has a highly typical age distribution. The classical description of this age pattern is by Comstock et al, who followed 82,269 Puerto Rican children reacting to tuberculin enrolled in 1949–1951 for 8 to 20 years [Figure 1]38. This pattern, confirmed in a systematic review of studies from the pre-chemotherapy era39, is defined by a peak in the first 1–4 years of life, followed by a trough until early puberty, rising to a second peak around the age of 20 years. Analyses of notification and prevalence data from high-incidence countries show that incidence starts to rise again from the sixth decade40,41. Although several explanations for this age pattern have been suggested, none has been proven.\n\nAverage annual rate of tuberculosis disease in a cohort of 82,269 Puerto Rican children with a positive tuberculin skin test, by age of disease occurrence. Children were enrolled in the period 1949–1951, and followed for 8 to 20 years. Figure reproduced with permission from Comstock et al. (1974)38.\n\nInfants. Studies from the pre-chemotherapy era showed that, while the risk of infection with Mtb in the first year of life was over 10-fold lower than later in childhood, the risk of progression to disease once infected was much higher with up to 50% of infected infants developing disease39. These high progression rates have been attributed to age-specific maturation of immune responses42, although the mechanisms responsible for this vulnerability have not been elucidated43.\n\nHCMV infection in infants is common44. Depending on the country and socio-economic status of the mother, between 10 and 60% of children are HCMV IgG seropositive (reflecting current or past active infection) by the age of 12–36 months45–52. Important causes are congenital infection and transmission through breastfeeding; >85% of HCMV seropositive women excrete virus in the breastmilk44,53–56. Infants infected through breastfeeding do not develop disease, probably due to protection by maternal antibodies, but do shed virus in saliva and urine intermittently for months, by which they may transmit HCMV to other children and caregivers23,44,48,57. Shedding of HCMV shows a steep decline by the age of 5 years23, coinciding with the age at which TB incidences drop38,39.\n\nAdolescents. The rate of progression to TB disease then remains low until puberty. Several studies have observed an increase in TB incidence from this age onward among children who were exposed to infectious TB patients or had a positive tuberculin response, leading to a peak in incidence in the first half of the third decade38,58–63. This phenomenon has been attributed to hormonal changes, but again without a putative mechanistic pathway64.\n\nMost population-based studies of HCMV seroprevalence show exactly this age pattern: a slow increase in HCMV IgG seroprevalence up to the age of 10–15 years, followed by an acceleration during adolescence45,47–50,52,65–72. One explanation for this increase in seroprevalence is sexual transmission. Various studies found that HCMV conversion among women was associated with sexual activity73–77. However, as several studies of adolescents found no association of HCMV seroprevalence with sexual exposure67,78,79, other transmission routes such as mouth-to-mouth kissing may also be important.\n\nAnother indication that HCMV infection may be implicated is the sex difference in TB disease progression in the second decade. For girls the increase in TB incidence starts 2–4 years earlier than for boys, and progression rates tend to remain higher in women than in men for the subsequent two decades, a pattern that was observed before the HIV era in various populations38,59,60,62,63,80–83. This pattern is again reflected in that of HCMV infection. The acceleration of HCMV seroprevalence during puberty and adolescence is steeper in girls than in boys and is higher in women of childbearing age than in men in populations with relatively low HCMV seroprevalence33,48,49,68,84–86. Age-adjusted HCMV seroprevalence does not differ between men and women in populations with high seroprevalence33,87. This may be because IgG seroprevalence measures cumulative infection experience and thus ignores reinfection. HCMV reinfection, identified by DNA typing or strain-specific antibody responses, is a common occurrence in sexually exposed women88–90.\n\nElderly. Although there is little data on TB progression rates in the elderly, age patterns of TB notifications suggest increased progression rates from the sixth decade onward1,40. In populations with declining incidence rates over the past decades this is partially a cohort effect, whereby younger generations have lower prevalence of latent infection91,92. However in high-incidence countries with little change in TB incidence, notification rates clearly increase at older age1. This is also observed for TB prevalence in population surveys, suggesting that this is not explained by better access to diagnosis1. HCMV infection has been implicated as a cause of age-related decrease in naïve T cells and increase in memory T cells known as immunosenescence93. However, reactivation of HCMV infection is also common at old age, probably reflecting weakening immune control29. Detection of viral DNA increases after the age of 60–70 years94,95, and viral DNA is frequently detected in urine and plasma of elderly people96,97.\n\nOur hypothesis predicts that factors that drive CMV (re-)infection are also risk factors for TB. We highlight the four most important: socio-economic status, sexual contact, blood transfusion, and solid organ transplantation.\n\nSocio-economic status. Incidence and prevalence of CMV infection are associated with poor socio-economic status (SES), between countries as well as within countries and communities33,48,49,84,98–100. This includes association with crowding, in particular the number of young children in household101–103. Several studies found ethnicity or migrant status to be independently associated with age-adjusted CMV prevalence49,68,104, which may partly reflect higher background infection rates in the country of origin. In a US study the association with ethnicity was explained by differences in exposure to infants and sexual risk77.\n\nAlso the incidence of TB, often regarded as the archetypal poverty disease, shows a remarkable inverse gradient with SES at the household, regional and country level105–107. This association has been explained mainly by crowding in ill-ventilated spaces conducive to Mtb transmission108, poor nutritional status12,109, alcohol abuse15 and, possibly, indoor air pollution110. Similarly, in low-incidence countries, TB incidences are higher in particular ethnic groups and immigrants111,112, which also may reflect socio-economic disparities and differences in background infection rates113. Very few studies have attempted to investigate whether these and other known risk factors explain all of the observed variation in SES-related TB incidence114.\n\nSexual contact. The risk of CMV (re-)infection in adults is correlated with measures of sexual activity such as age at first intercourse, recent and lifetime number of sexual partners and condom use, as well as with prevalence of other sexually transmitted infections73,74,76,115–117. Historically, TB has also been associated with sexual promiscuity in medical and popular literature (reviewed in 37) but no systematic epidemiological data exist. Investigation of associations between TB disease and sexually transmitted infections has been strongly dominated by HIV infection, which may obviously be a major confounder. There have been few studies from low HIV prevalence populations. One from China found an association between history of TB and human papilloma virus infection118.\n\nInterestingly, the declining TB mortality rates in The Netherlands and England and Wales in the 20th century showed no surge during the Great Depression105,119, when SES status deteriorated thereby affecting several of these known risk factors, in particular nutritional status. They did however surge during and shortly after the Second World War105,119. In England and Wales this was not paralleled by major deterioration in nutritional status; in The Netherlands famine only started in the winter of 1944–45 while the increase in TB mortality started already from 194237. In both countries during this period major increases were seen in sexually transmitted infections, mainly related to presence of large numbers of Allied and Axis troops37.\n\nBlood transfusion. Transfusion-associated CMV infection occurs in particular following multiple transfusions of whole blood or granulocytes, and can be prevented by removal of white blood cells120–122. Increased incidences of TB have indeed been described in two categories of patients who in the past often received multiple whole blood transfusions: patients who underwent (partial) gastrectomy, mainly for bleeding gastric ulcers123,124, and patients with end-stage renal disease on haemodialysis125. For both these categories alternative explanations for increased TB incidences are possible: low body mass index for gastrectomy123,124, and impaired cellular immunity due to uraemia for haemodialysis126. Nonetheless, several studies among haemodialysis patients have suggested increased rates of CMV (re)infection, either or not associated with transfusion of blood or blood products127–131, as well as increased rates of CMV reactivation127.\n\nSolid organ transplantation. The incidence of symptomatic CMV infection is strongly increased in solid organ transplant patients, mainly due to infection from a CMV IgG positive donor132. Solid organ transplantation also increases the risk of TB disease125,133–135. TB incidence is highest in lung transplant patients and associated with presence of latent TB infection, clinical condition and intensity of the immunosuppressive therapy; the latter has been brought forward as the sole explanation for the increased TB risk136. Interestingly, a study among Korean solid organ transplant patients found that the risk of developing TB was associated with CMV infection within the prior 3 months137.\n\n\nPotential impact on tuberculosis control and elimination\n\nIf indeed CMV (re-)infection or reactivation commonly precipitates progression from latent infection to active TB, this will suggest novel approaches to TB control. Combining a test for Mtb infection with one for ongoing or recent active CMV infection may strongly increase our ability to predict the development of TB disease and allow the targeting of preventive treatment to those most at risk4. Vaccination against CMV might prevent TB in those with TB infection. A wide range of CMV vaccines are currently in clinical development including plasmid-based vaccines, viral vector vaccines, attenuated HCMV strains, and recombinant protein and peptide vaccines138. Recently, a genetically modified CMV vector expressing antigens from Mtb (RhCMV/TB) has shown 41% sterile protection against Mtb in a non-human primate study139. If the human version of this vaccine was able to afford (partial) protection against CMV, it could also significantly impact the TB epidemic. CMV infection may also affect TB treatment response, another potential application therefore could be the provision of CMV antiviral treatment as an adjunct to TB treatment, for example of patients with multidrug resistance.\n\n\nFuture research\n\nThere is an urgent need for elucidating the role of CMV infection in TB disease progression. Further serological and cellular studies should be done to confirm the association between TB disease and CMV infection. However, in settings with high CMV seroprevalence (also those with highest TB incidence) it will be important to identify recent reinfection for which various diagnostic approaches exist22. Their relative merits are beyond the scope of this article, but some may potentially signal reactivation due to Mtb replication, i.e. consequence rather than cause. Therefore, ultimately longitudinal studies are needed in which the incidence of TB disease among those with latent TB infection is measured over time comparing those with CMV (re-)infection or reactivation to those without. These studies should be supplemented with immunological studies to define the mechanisms through which CMV precipitates progression to active TB disease. Finally, it will be important to study the role of CMV reactivation during TB disease and its effect on the response to TB treatment.", "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\nWHO \| Global tuberculosis report 2017. WHO: Geneva, 2017. Reference Source\n\nFerebee SH: Controlled chemoprophylaxis trials in tuberculosis. A general review. Bibl Tuberc. 1970; 26: 28–106. PubMed Abstract\n\nDheda K, Barry CE 3rd, Maartens G: Tuberculosis. 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Drugs. 2016; 76(17): 1625–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHansen SG, Zak DE, Xu G, et al.: Prevention of tuberculosis in rhesus macaques by a cytomegalovirus-based vaccine. Nat Med. 2018; 24(2): 130–43. PubMed Abstract \| Publisher Full Text" }	[ { "id": "31552", "date": "14 Mar 2018", "name": "Blair L. Strang", "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 interaction of different pathogens in an infected individual is a fascinating subject, but, in general, poorly understood. For the most part, it is thought that infection with one pathogen facilities the replication and pathogenesis of another. An example of this is the clear and long standing link between human immunodeficiency virus (HIV) infection and development of severe disease related to MTB mycobacterium tuberculosis (MTB) infection. What is interesting of MTB infection are the concepts of predisposing or preexisting infection other than HIV linked to MTB (for example, the widespread respiratory pathogen influenza.)\n\nIn this article, the authors outline intriguing lines of converging evidence detailing the possible association of human cytomegalovirus (HCMV) and MTB infection, suggesting that HCMV infection is a relevant factor in the incidence of MTB disease.\n\nThe evidence presented rests largely on epidemiological findings involving age distribution and congruent risk factors. There is an interesting link between the incidence of MTB in early life and adolescence that mirrors acquisition of HCMV either during pregnancy/post partum or by sexual contact in adolescence. Several congruent risk factors are discussed. However, the authors concede that these factors could be applicable to many widespread opportunistic pathogens, including HCMV.\n\nOverall, the argument presented based on epidemiological evidence is compelling and the possible link between HCMV and MTB infection should be investigated further. Indeed, the authors should have done more to highlight very resent work from one of their laboratories on this topic. This study, mentioned in the opening paragraphs, shows a clear association of the presence of T cells recognizing HCMV and an increased risk of MTB disease early in life. What is especially interesting about this study is the possibility that development of MTB disease is related to the impairment of NK cell function. Much recent work in HCMV pathogenesis by Wilkinson and colleagues has highlighted the many and diverse mechanisms that HCMV employs to evade NK cells. It is interesting to consider that HCMV evasion of the immune response to infection is directly related to development of MTB disease.\n\nOther points surrounding mechanisms linking HCMV and MTB infection are also unclear. The authors mention that the lung may be a reservoir for HCMV. However, this is based on studies in mice using murine cytomegalovirus (MCMV). The pathology of MCMV does not accurately mirror that of HCMV. Thus, more compelling evidence relating the presence of HCMV in the lung with disease must be presented. Based on the data discussed above, co-incident infection of HCMV and MTB in lung tissue may not be required for disease, but, perhaps, modulation of the immune system.\n\nFinally, the authors consider therapeutic intervention. They highlight the sterling work of Picker and colleagues using rhesus cytomegalovirus (RhCMV) as a vaccine vector. These studies have shown that in non-human primates a RhCMV-MTB vaccine candidate can offer notable protection against challenge with MTB. It must be noted, however, that there is currently no widely available HCMV vaccine and it is unclear if vaccines based on HCMV in humans will offer similar protection to those based on RhCMV in non-human primates. It may be some time before viable HCMV based vaccines are available. In the short term it may be better to peruse the other suggestion the authors make, which is to trial the use of anti-HCMV drugs in patients with MTB.\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": "3608", "date": "30 Apr 2018", "name": "Frank Cobelens", "role": "Author Response", "response": "We thank Dr. Strang for his valuable comments.Here we respond point-by-point, referring to the revised version of our paper.1. \"(... )Indeed, the authors should have done more to highlight very resent work from one of their laboratories on this topic. This study, mentioned in the opening paragraphs, shows a clear association of the presence of T cells recognizing HCMV and an increased risk of MTB disease early in life. What is especially interesting about this study is the possibility that development of MTB disease is related to the impairment of NK cell function. Much recent work in HCMV pathogenesis by Wilkinson and colleagues has highlighted the many and diverse mechanisms that HCMV employs to evade NK cells. It is interesting to consider that HCMV evasion of the immune response to infection is directly related to development of MTB disease.\"Response: We agree that the possible role of impairment of NK cell function deserves more attention. We now elaborate on this aspect in the section entitled \"Human Cytomegalovirus Infection\".2. \"The authors mention that the lung may be a reservoir for HCMV. However, this is based on studies in mice using murine cytomegalovirus (MCMV). The pathology of MCMV does not accurately mirror that of HCMV. Thus, more compelling evidence relating the presence of HCMV in the lung with disease must be presented. Based on the data discussed above, co-incident infection of HCMV and MTB in lung tissue may not be required for disease, but, perhaps, modulation of the immune system.\"Response: We added the following references: Gordon et al, J Exp Med 2017 (a study of donor organs that showed the lung is a major site for CMV DNA) and Poole et al. J Infect Dis 2015 (that found CMV in alvealoar macrophages). But it is also true that CMV may not need to be present in the lung as it impacts the systemic immune response and therefore has an indirect effect still relevant for lung immunity. It is possible that both co-infection of lung AND CMV impact on systemic immune response contribute to TB risk in the CMV infected individual. We added this consideration to the in the section entitled \"Human Cytomegalovirus Infection\".3. \"It must be noted, however, that there is currently no widely available HCMV vaccine and it is unclear if vaccines based on HCMV in humans will offer similar protection to those based on RhCMV in non-human primates.\" Response: We agree with this caveat that we added to the text." } ] }, { "id": "32121", "date": "22 Mar 2018", "name": "Maziar Divangahi", "expertise": [ "Reviewer Expertise TB Immunologist" ], "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\nOur continuous exposure to a multitude of pathogens requires a greater understanding of the host response to heterologous infections and is fundamental for developing novel therapies and vaccines against infectious diseases. In line with this, heterologous immunity to viruses and bacteria is an extremely complex scenario as our immune response greatly differs upon viral versus bacterial infection. Thus, how our immune system makes a balanced response to control both viral and bacterial infections without killing the host is a fascinating area of research.\n\nIn this review article, Cobelens and colleagues reviewed the available epidemiological data for human cytomegalovirus (HCMV) and Mycobacterium tuberculosis (Mtb) infections. The review is well written and provides interesting human observations that HCMV may contribute to the progression of latent TB to active disease. However, I have three major comments that potentially can improve the conceptual idea of the current review.\n\n1) Type I IFN is a chief antiviral cytokine, which also plays a critical role in immunity to CMV infection (Plos Pathogens, 10:e1003962, 2014). However, it has been well documented that type I IFN increases host susceptibility to some bacterial infections including Francisella tularensis (J. Immunol. 169, 1665-1668, 2002), Listeria monocytogenes (J. Exp. Med. 185:921, 1997) and Mtb (J. Interferon Cytokine Res. 25:694, 2005 & Nature, 511:99, 2014). This concept has been also supported by another recent study (JID, 290:270 2014) demonstrating influenza infection increased susceptibility to Mtb infection in a Type I IFN dependent manner. Although the authors briefly mention this idea, a cohesive expansion of this concept would provide further insight for the important role of type I IFN in viral infection and susceptibility to Mtb (Trends in Immunology, 36:307, 2015).\n\n2) The authors need to be cautious about their conclusions regarding a recent paper using nonhuman primate (rhesus macaques) cytomegalovirus vectors encoding Mtb antigens (RhCMV/MTB) (Nature Medicine, 24:130, 2018) for the vaccine in TB. Although the protective data from this study are very impressive, the lack of cellular mechanism (e.g. the protection was independent of effector T cells) as well as the lack of several important controls including the empty vector (RhCMV) indicate that further investigations are required to have a better understanding of this approach as a vaccine in pre-clinical study prior to any human trial for TB.\n\n3) The presented human observations that sexual contact and whole blood transfusion are the risk factors for tuberculosis is very speculative. Thus, the author should consider rephrasing this section at least in the abstract as it sounds very confirmative.\n\nMinor comments:\n\n1) The Age-specific death rates from TB in England and Wales was also reported for 1913 and 1918 that shows the same pattern (Table 2) and the authors may want to include this reference:\n\nLangford, C. The age pattern of mortality in the 1918–1919 influenza pandemic: an attempted explanation based on data for England and Wales. Med. Hist. 46, 1–20 (2002).\n2) As there is no evidence of Mtb activating latent HCMV in the lungs (Page 3), which is also not the focus of this review, the author may want to consider removing this entire paragraph.\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? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "3607", "date": "30 Apr 2018", "name": "Frank Cobelens", "role": "Author Response", "response": "We thank Dr. Divangahi for his valuable comments.Here we respond point-by-point, referring to the revised version of our paper.Major comments1: \"(...) Although the authors briefly mention this idea, a cohesive expansion of this concept would provide further insight for the important role of type I IFN in viral infection and susceptibility to Mtb\". Response: The reviewer is right to point out that Type I IFN plays a role in HCMV infection as well as in Mtb infection. We expanded the paragraph on viral triggers to include more detail on the possible role of Type I IFN.2. \"The authors need to be cautious about their conclusions regarding a recent paper using nonhuman primate (rhesus macaques) cytomegalovirus vectors encoding Mtb antigens (RhCMV/MTB) (Nature Medicine, 24:130, 2018) for the vaccine in TB. (...)\"Response: We agree with this criticism and now phrased this more cautiously by pointing out that there is currently no widely available HCMV vaccine and it is unclear if vaccines based on HCMV in humans will offer similar protection to those based on RhCMV in non-human primates.3. \"The presented human observations that sexual contact and whole blood transfusion are the risk factors for tuberculosis is very speculative. Thus, the author should consider rephrasing this section at least in the abstract as it sounds very confirmative.\" Response: We agree that for these two risk factors the evidence base is limited, although we do point out reasons why (e.g. the confounding effect between TB and sexual contact by HIV status). We rephrased this statement in the abstract as \"(...) including poor socio-economic status, solid organ transplantation and, possibly, sexual contact and whole blood transfusion.\".Minor comments1. \"The age-specific death rates from TB in England and Wales was also reported for 1913 and 1918 that shows the same pattern (Table 2) and the authors may want to include this reference:Langford, C. The age pattern of mortality in the 1918–1919 influenza pandemic: an attempted explanation based on data for England and Wales. Med. Hist. 46, 1–20 (2002).\"Response: Indeed TB mortality showed a peak in 1918 in England and Wales, as it did in Switzerland and South Africa (as referenced), as well as in other countries such as The Netherlands (not referenced). This mortality peak has generally been associated with the Spanish Flu epidemic rather than with WW-I, among other because several of these countries were not involved in the war. We feel that this point is rather secondary to our reasoning and does not need additional referencing. Note that mortality peak in 1913 observed by Langford in in England and Wales was observed only in the age group 0-4 years, with little bearing on overall mortality.In light of this discussion we feel it is important to point out that increased TB mortality during influenza epidemics may reflect increased case fatality among TB patients due to secondary influenza rather than increased TB incidence. We now added this consideration to the paragraph on viral triggers of TB.2. \"As there is no evidence of Mtb activating latent HCMV in the lungs (Page 3), which is also not the focus of this review, the author may want to consider removing this entire paragraph.\"Response: We agree that this aspect is outside the focus of our review and took out the part of the sentence related to Mtb infection reactivating HCMV infection. We did leave in the remainder of the paragraph but did include a statement that the effect of HCMV on the immunity to Mtb may be systematic as well as local, i.e. in the lungs." } ] } ]	1	https://f1000research.com/articles/7-280
https://f1000research.com/articles/7-512/v1	27 Apr 18	{ "type": "Research Note", "title": "Bioethanol fermentation from kitchen waste using Saccharomyces cerevisiae", "authors": [ "Shafkat Shamim Rahman", "Md. Mahboob Hossain", "Naiyyum Choudhury", "Md. Mahboob Hossain", "Naiyyum Choudhury" ], "abstract": "Bioethanol obtained from microbial fermentation can replace conventional fossil fuels to satisfy energy demand. In this respect, a fermenting isolate of Saccharomyces cerevisiae, obtained from date juice, was grown in YEPD medium as a part of a previous published research project. In this study, the isolate was tentatively characterized for alcoholic fermentation in organic kitchen waste medium, prepared from discarded fruit and vegetable peels. Fermentation in shaking condition resulted in the production of 7.3% (v/v) ethanol after 48 h, after which the pH of the medium increased slightly in response. Further research should be conducted to assess the potential of kitchen waste as a raw material in ethanol fermentation.", "keywords": [ "Kitchen waste", "Saccharomyces cerevisiae", "Fermentation", "Bioethanol" ], "content": "Introduction\n\nKitchen waste is a raw material available in large volumes. The term applies to organic solids, which are discarded during food preparation. Kitchen waste is mostly composed of lignocelluloses and starch, and is degradable through microbial infestation. An outstanding resource for biotechnology is present in kitchen waste as carbohydrate polymer fraction1. The usage of lignocelluloses as feedstock would prompt novel challenges for biotechnology, for example, the product diversification2,3.\n\nKitchen waste extracted bioethanol is an alluring and sustainable energy source for vehicle fuel, as a gasoline alternative. Present ethanol production (so-called ‘first generation’), utilizing harvests such as sugar cane and corn, has become conventional method, whereas second-generation ethanol generation uses less expensive, non-sustenance feedstocks, for example, lignocelluloses or municipal solid waste, which could make ethanol more competitive alternative to petroleum4,5.\n\nPlant cell walls are mostly composed of lignocellulosic biomass. Among its main components is cellulose, a linear polymer of cellobiose consisting of two D-glucose molecules connected by β-1, 4 bonds6. The more organized or crystalline cellulose is, the less soluble and less degradable it is. Cellulose degradation techniques include the use of effective enzymes, concentrated acid or alkaline and high temperatures for both nebulous and crystalline cellulose in the transformation procedure. Cellulose would be an ideal carbohydrate source for the fermentation due to the uniform hydrolyzable glucose building blocks7.\n\nHemicellulose is another important hetero-polysaccharide present in the plant cell wall. Hemicellulose differs from cellulose by the organization of several sugar units, by the presence of shorter chains and by a ramified central chain. Hemicellulose removal from the plant cell wall is easier than lignin and cellulose, owing to the bonds between cellulose, hemicellulose and lignin. A wide variety of enzymes, including endoxylanase, exoxylanase, mannanase, arabinosidase, acetylesterase, and glucoronisidase, are essential due to its structural diversity8. Alkaline pretreatments were also found to be successful at degrading hemicellulose9.\n\nLignin provides additional strength and protection to prevent enzymatic activity of fungi and insect attack by linking cellulose and hemicellulose10. Substantial moisture and rigidity resistance also added to biomass11 and known as a cellulase inhibitor12. As a result, exogenous proteins, such as bovine serum albumin, and surfactants, such as MgSO4, and CaCl213, are added before microbial or enzyme loading. Moreover, many pretreatment processes have been established to moderate the lignin hindrance.\n\nFunctional groups of lignin, including phenolic hydroxyl, benzyl hydroxyl, methoxyl, carbonyl and a minor amount of terminal aldehyde groups, are factors that influence its decomposition14. The lignin carbohydrate complexes (syringyl, guaiacyl and p-hydroxyphenyl units) formed by crosslink interactions, are also counted as a fermentation-restricting factor.\n\nStarch (α-D-glucose monomer) degradation is complex than the sugar fermentation process. It initially broke down into glucose, through amylase or diastase and maltase hydrolysis. Then, ethanol and carbon dioxide are fermented from sugars through enzyme activity.\n\nAlongside a mainstream project15,16, in this study, tentative fermentation was carried out in kitchen waste medium to find out if a wild-type microorganism has the ability to ferment cellulose efficiently17.\n\n\nMethods\n\nYeast samples (Saccharomyces cerevisiae) were isolated and identified from date-juice using previously described methods15,18 in YEPD medium (106–107 cells/ml; 0.3% yeast extract #Y1625, 1% peptone #P7750, 2% dextrose #G8270, 1.5% agar #A1296, pH: 5; Sigma-Aldrich, St Louis, MO, USA). Discarded solid kitchen waste was collected from different households. This included peels from potatoes, pumpkin, papaya, cucumber, okra, green banana, balsan apple, carrot and basil. After chopping, pulverizing and blending with 1 L water, 250 g solid waste was taken as raw medium. Concentrated HCl (2 ml) was added to convert the calcium present (a fermentation inhibitor) to calcium sulfate salt19. HCl also regulates the pH of the medium to control for bacterial contamination and facilitate chemical hydrolysis of plant residues, which were boiled for 1.5 h, giving carbohydrate units of cellulose and starch. Monomers of amylose, amylopectin and glucose arose from further degradation. Urea (0.1 g) was also supplemented prior to boiling as a nitrogen source nutrient. The final pH was adjusted to 6.0 by dropwise addition of NaOH or HCl (measured using a pH meter; Mettler Toledo, Switzerland).\n\nThe 250 ml fermentation medium was transferred into 500 ml Erlenmeyer flasks and a homogenous suspension of yeast (10 ml YEPD broth) was inoculated in aseptic conditions. The flask was incubated in a rotary incubator (120 rpm) at 30°C for 48 h. Two separate experiments were conducted and ethanol production was recorded at 24 and 48 h intervals, and the average were calculated. The ethanol in this experiment was analyzed using the Conway method20. Downstream processing is required before isolation of usable ethanol.\n\n\nResults\n\nPrevious investigations15,16,18 indicated that ethanol is produced more readily under shaking than non-shaking conditions. After 48 h of fermentation at room temperature in a rotary incubator (120 rpm), a maximum of 7.3% (v/v) ethanol production was recorded (Table 1). The rate of alcohol production showed a cumulatively increasing trend, which was mirrored by a continued rise in pH throughout incubation, recorded as pH 6.52 at 48 h. The results also indicated that the full potential of kitchen waste fermentation will be revealed through longer durations of fermentation.\n\n\nDiscussion\n\nThe kitchen waste medium contained plant organelles and was a rich source of cellulose, starch and glucose monomers. Comparison with previous studies15,16,18 showed the achieved production efficiency is below the level required for profitable commercial production. In this study, production of 7.3% (v/v) ethanol was recorded (Table 1). Further optimization of the process and co-fermentation (e.g. ethanol–butanol co-fermentation) is among the future goals of researchers21.\n\nMost of the kitchen waste was similar feedstock to lignocellulosic raw materials, which is considered to be an excellent substrate22. Previous works delineated the pathway of converting plant-based waste biomass to bioethanol, in which enzyme pretreatment was conducted before yeast fermentation23–26. Gnansounou & Dauriat produced 30.9 g bioethanol and 65.2 L biogas using 1 kg of kitchen waste27. Velasquez & Ruiz fermented 346.5–388.7 l/ton bioethanol in a similar study using banana pulp and skin28. Industrial waste has also been used in fermentation technology29. A planned facility in East London will produce 16 million gallons of jet fuel per annum from 500,000 tons of waste for British Airways (the Green Sky project). A combination of plasma arc gasification with the Fischer-Tropsch method, known as Solena's Plasma Gasification (SPG) technology, will be used. In China’s Jiangsu province, a new waste-to-energy facility with a processing capacity of 900 metric tons, will be constructed.\n\n\nConclusions\n\nResults were derived from limited parameters and only a single isolate of microorganism was employed. Future studies should be directed towards elaborate characterization and compare different criteria of kitchen waste fermentation. Optimization of the media and physicochemical parameters, and longer-duration fermentation will also be performed in future. This study was aimed towards fermentation only. A cheap, efficacious downstream processing method of the ethanol generated in this process also requires development.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nAuthor information\n\nWhen the research was carried out, SSR was a MSc student at Biotechnology program, Department of Mathematics and Natural Sciences, BRAC University and NC was the Coordinator of Biotechnology and Microbiology programmes at the Department of Mathematics and Natural Sciences, BRAC University.", "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 are grateful to the late Prof. Dr. AAZ Ahmad, former Chairperson of Department of MNS, BRAC University, Dhaka, for allowing the research work. Their sincere appreciation goes to Armanul Nasir, Arif, Forkan and Shamim Akhter Chowdhury for their graceful help and support.\n\n\nReferences\n\nHossain N, Zaini JH, Mahlia TM: A Review of Bioethanol Production from Plant-based Waste Biomass by Yeast Fermentation. Int J Technol. 2017; 8(1): 5–18. Publisher Full Text\n\nPeris D, Moriarty RV, Alexander WG, et al.: Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production. Biotechnol Biofuels. 2017; 10: 78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJayakody LN, Ferdouse J, Hayashi N, et al.: Identification and detoxification of glycolaldehyde, an unattended bioethanol fermentation inhibitor. Crit Rev Biotechnol. 2017; 37(2): 177–89. PubMed Abstract \| Publisher Full Text\n\nLee YG, Jin YS, Cha YL, et al.: Bioethanol production from cellulosic hydrolysates by engineered industrial Saccharomyces cerevisiae. Bioresour Technol. 2017; 228: 355–61. PubMed Abstract \| Publisher Full Text\n\nLee CR, Sung BH, Lim KM, et al.: Co-fermentation using Recombinant Saccharomyces cerevisiae Yeast Strains Hyper-secreting Different Cellulases for the Production of Cellulosic Bioethanol. Sci Rep. 2017; 7(1): 4428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLynd LR, Weimer PJ, van Zyl WH, et al.: Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev. 2002; 66(3): 506–577. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang YH, Lynd LR: Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol Bioeng. 2004; 88(7): 797–824. PubMed Abstract \| Publisher Full Text\n\nThygesen A, Thomsen AB, Schmidt AS, et al.: Production of cellulose and hemicellulose-degrading enzymes by filamentous fungi cultivated on wet-oxidised wheat straw. Enzyme Microb Technol. 2003; 32(5): 606–15. Publisher Full Text\n\nAlvira P, Tomás-Pejó E, Ballesteros MJ, et al.: Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review. Bioresour Technol. 2010; 101(13): 4851–61. PubMed Abstract \| Publisher Full Text\n\nHoward RL, Abotsi E, Van Rensburg ELJ, et al.: Lignocellulose biotechnology: Issues of bioconversion and enzyme production. Afr J Biotechnol. 2003; 2(12): 602–619. Publisher Full Text\n\nChandra RP, Bura R, Mabee WE, et al.: Substrate pretreatment: the key to effective enzymatic hydrolysis of lignocellulosics? Adv Biochem Eng Biotechnol. 2007; 108: 67–93. PubMed Abstract \| Publisher Full Text\n\nMansfield SD, Mooney C, Saddler JN: Substrate and Enzyme Characteristics that Limit Cellulose Hydrolysis. Biotechnol Prog. 1999; 15(5): 804–16. PubMed Abstract \| Publisher Full Text\n\nLiu H, Zhu JY, Fu SY: Effects of lignin-metal complexation on enzymatic hydrolysis of cellulose. J Agric Food Chem. 2010; 58(12): 7233–8. PubMed Abstract \| Publisher Full Text\n\nHiguchi T: Lignin Biochemistry: Biosynthesis and Biodegradation. Wood Sci Technol. 1990; 24(1): 23–63. Publisher Full Text\n\nRahman SS, Hossain MM, Choudhury N: Effect of Various Parameters on the Growth and Ethanol Production by Yeasts Isolated from Natural Sources. Bangladesh J Microbiol. 2013; 30(1–2): 49–54. Publisher Full Text\n\nNasir A, Rahman SS, Hossain MM, et al.: Isolation of Saccharomyces Cerevisiae from Pineapple and Orange and Study of Metal’s Effectiveness on Ethanol Production. Eur J Microbiol Immunol (Bp). 2017; 7(1): 76–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang J, Hu M, Zhang H, et al.: Converting Chemical Oxygen Demand (COD) of Cellulosic Ethanol Fermentation Wastewater into Microbial Lipid by Oleaginous Yeast Trichosporon cutaneum. Appl Biochem Biotechnol. 2017; 182(3): 1121–1130. PubMed Abstract \| Publisher Full Text\n\nRahman SS, Sarkar MKI, Islam MR, et al.: Isolation of yeasts from raisins and palm-juice and ethanol production in molasses medium. Indian J Sci Technol. 2016; 9(12). Publisher Full Text\n\nTaherzadeh MJ, Karimi K: Process for ethanol from lignocellulosic materials I: Acid-based hydrolysis processes. BioResources. 2007; 2: 472–499. Reference Source\n\nObrink KJ: A modified Conway unit for microdiffusion analysis. Biochem J. 1955; 59(1): 134–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi YJ, Lu YY, Zhang ZJ, et al.: Co-fermentation of Cellulose and Sucrose/Xylose by Engineered Yeasts for Bioethanol Production. Energy & Fuels. 2017; 31(4): 4061–7. Publisher Full Text\n\nkabashi NA, Alam Z, Ainuddin M: Bio-composting Process Development by SSF for Utilization Agro-industrial Wastes. In: IFMBE Proceedings. 2007; 15: 464–468. Publisher Full Text\n\nFranceschin G, Sudiro M, Ingram T, et al.: Conversion of Rye Straw into Fuel and Xyitol: a Technical and Economical Assessment Based on Experimental Data. Chemical Engineering Research and Design. 2011; 89(6): 631–640. Publisher Full Text\n\nHossain N, Jalil R: Sugar and Bioethanol Production from Oil Palm Trunk (OPT). Asia Pacific Journal of Energy and Environment (APJEE). 2015; 2: 89–92. Reference Source\n\nCasa-Villegas M, Marín-Navarro J, Polaina J: Synergies in coupled hydrolysis and fermentation of cellulose using a Trichoderma reesei enzyme preparation and a recombinant Saccharomyces cerevisiae strain. World J Microbiol Biotechnol. 2017; 33(7): 140. PubMed Abstract \| Publisher Full Text\n\nYamada R, Nakashima K, Asai-Nakashima N, et al.: Direct Ethanol Production from Ionic Liquid-Pretreated Lignocellulosic Biomass by Cellulase-Displaying Yeasts. Appl Biochem Biotechnol. 2017; 182(1): 229–37. PubMed Abstract \| Publisher Full Text\n\nGnansounou E, Dauriat A: Ethanol Fuel from Biomass: A Review. J Sci Ind Res. 2005; 64: 809–821. Reference Source\n\nVelásquez-Arredondo HI, Ruiz-Colorado AA, De Oliveira junior S: Ethanol production process from banana fruit and its lignocellulosic residues: Energy analysis. Energy. 2010; 35(7): 3081–3087. Publisher Full Text\n\nPark L, Kim L, Kang K, et al.: Cellulose Ethanol Production from Waste Newsprint by Simultaneous Saccharification and Fermentation using Saccharomyces cerevisiae KNU5377. Process Biochemistry. 2010; 45(4): 487–492. Publisher Full Text" }	[ { "id": "33555", "date": "19 Jun 2018", "name": "Parveen Fatemeh Rupani", "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 manuscript has lack of novelty in term of methodology procedure.\nThe introduction I suggest to focus more on the previous studies of generating ethanol from kitchen waste and show the gap in between. Clear the objective of the study.\nThe methodology has not been explained well. The process was under anaerobic or aerobic condition? What was the pH selected for the experiment? No explanation of the pre-treatment of the material, the initial composition characteristics. Also authors did not mention about the size of the sample taken.\nAuthors have not describe the analytical method used in the study. Full description of the equipment and the methods required.\nAuthors have to explain in details how many samples were examined, how may runs. And also provide the statistical data in order to discuss the significances of the results.\nThe results and discussion is very weak and only presented one table. Which is not in scientific form. Discussion should be with comparison of the previous studies.\nTherefore, I do not recommend for indexing.\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? No\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": "35936", "date": "30 Aug 2018", "name": "Mohidus Samad Khan", "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 current version of the submitted article doesn’t have sufficient content and merit to be indexed as a full journal article. The storyline of the article is not well developed, the relevant references are not well cited, the article doesn’t have sufficient results for critical analysis (and how these results are different from other reported results, unless those results are unique and reported for the first time).\nIt can be included in a conference proceedings (or may not); however, for journal articles we expect high standard research work and publication.\n\nThis article reports the production of bioethanol from kitchen waste fermentation.\n\nNot technically sound.\n\nThe article does not add any new information to the scientific community.\n\nVery limited results.\n\nNo comparison/report on similar work around the world.\n\nThis article is not suitable for indexing.\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? Partly\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? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/7-512
https://f1000research.com/articles/7-507/v1	27 Apr 18	{ "type": "Method Article", "title": "An inexpensive and easy-to-make customized antibiotics mix for mycobacterium culture", "authors": [ "Ashwani Kesarwani", "Puja Nagpal", "Alaknanda Mishra", "Rana Zaidi", "Pramod Upadhyay", "Ashwani Kesarwani", "Puja Nagpal", "Alaknanda Mishra" ], "abstract": "The cultivation of mycobacteria often requires the use of several antibiotics to limit the growth of other rapidly growing micro-flora present in the growth medium. This antibiotic cocktail is one of the most expensive reagents required for mycobacterium culture. Here we present a customized antibiotics mix that is easy to prepare at a fraction of the cost of the commercially available antibiotic mixture that protects against transient flora, which are normally present in lungs, without affecting mycobacterial colony number.", "keywords": [ "mycobacterium culture", "antibiotics", "customized antibiotics mix" ], "content": "Introduction\n\nMycobacteria are slow-growing organisms (Lambrecht et al., 1988); to obtain visible colonies their culture has to continue for several days. Often the fluid to be examined for mycobacterium contains many other microflora and growth of this microflora has to be limited to allow the mycobacterium to grow. For this purpose, a cocktail of antibiotics is used in the culturing of mycobacterium. This antibiotics cocktail is one of the most expensive reagents (INR 4500 per pack, sufficient for 3 l of media) required for mycobacteria culture. We have formulated a Customized Antibiotics Mix (CAM), which is a mixture of antibiotics, to inhibit or reduce the growth of other micro-organisms.\n\nThe CAM has the following antibiotic components. Polymyxin B is a mix of polymyxin B1 and B2, basic polypeptides obtained from strains of Bacillus polymyxa. Polymyxin B acts as a bactericidal against all Gram-negative bacilli except Proteus and Neisseria genera by binding with the cell membrane and increasing its permeability, changing its structure and causing a higher uptake of water, ultimately leading to cell death (Cardoso et al., 2007).\n\nAmphotericin B is an antifungal drug first prepared from Streptomyces nodosus in 1955 (Donovick et al., 1955). This drug is also used to treat aspergillosis, blastomycosis, coccidioidomycosis, cryptococcosis and candidiasis. Amphotericin B causes the fungal cell to leak monovalent ions by binding with ergosterol, an integral part of fungal cell membrane, eventually causing fungal cell death (Mesa-Arango et al., 2012; O’Keeffe et al., 2003).\n\nNalidixic acid is a very weak organic acid used for the treatment of bacterial urinary tract infection such as Escherichia coli, Enterobacter, Klebsiella, Proteus and Shigella. It is a synthetic quinolone antibiotic, which is a group of antibiotics that inhibit bacterial growth by selectively blocking the DNA replication of these bacteria. Hence, it is also used for the study of regulation of bacterial division (Pommier et al., 2010).\n\nTrimethoprim is a synthetic antibacterial drug mainly used for the treatment of bladder infections. It typically targets species such as Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Enterobacter species. This drug inhibits the DNA synthesis of bacteria, hindering the reduction of dihydrofolic acid to tetrahydrofolic acid, which is a key precursor in the thymidine kinase pathway (Brogden et al., 1982).\n\nAzlocilin is a semisynthetic broad-spectrum antibiotic used against a number of Gram-positive and -negative bacteria. Azlocilin weakens the cell wall of the bacteria by binding to the penicillin-binding protein located inside the bacterial cell wall, which in turn inhibit the crosslinking of peptidoglycan (Sanders, 1983).\n\nAll of these aforementioned reagents are among those commonly used in antibiotics formulations for treating infections.\n\n\nMethods\n\nFor the preparation of CAM, the aforementioned commercially available antibiotic formulations were procured and their working stocks were prepared in water. From the working stock, the calculated amount volume of antibiotics were mixed. The details for the preparation of working stock and the final volume used for the preparation of 5 l Middlebrook 7H11 agar medium, sufficient for around 200 culture plates, are given in Table 1.\n\n*65 INR ≈ 1 USD.\n\nTypically, one tablet each of nalidixic acid (GramoNeg®; Best laboratories Pvt. Ltd.) and Trimethoprim (Bactrim®; Piramal Enterprises Limited) tablets were dispersed in 10 ml water and kept on rocker shaker for 15–20 mins, the mixture was then centrifuged at 600g for 10 min. Supernatant was aspirated and used for the preparation of the CAM.\n\nRequired amounts (shown in Table 1) of Polymyxin B (POLY-BTM; Samarth Life Sciences Pvt. Ltd.), Amphotericin B (AMPHOTRETTM; Bharat Serum and Vaccine Limited) and Azlocilin (Azenam; ARISTO Pharmaceuticals Pvt. Ltd.) were weighed and dissolved in water. The CAM was prepared by mixing appropriate volumes of working stock solutions and was filtered through a 0.2-µm syringe filter.\n\nA total of 105 g DifcoTM Mycobacteria 7H11 Agar (BD Biosciences, USA) was suspended in 4,500 ml water containing 25 ml glycerol. The medium was swirled on a hot magnetic plate to obtain a smooth suspension and autoclaved at 121°C for 15 min.\n\nThe medium was allowed to cool to 50–55°C in aseptic conditions. In the meantime, 25 g bovine serum albumin, 10 g dextrose, 15 mg catalase and 4.25 g sodium chloride (all Himedia, India) were dissolved in 500 ml water and filtered through a 0.2-µm filter. Next, 250 µl oleic acid (Himedia, India) was then added aseptically. This mix is commonly known as OADC (oleic acid, albumin, dextrose and catalase).\n\nTen vials of commercial BBLTM MGITTM PANTATM (BD-Panta) antibiotic mixture (Becton, Dickson and company, USA) was then added to 5 l media. In another preparation of media, the BBLTM MGITTM PANTATM was replaced with the CAM, formulated as aforementioned.\n\nTo compare the efficacy of the two antibiotic mixes, eight female B57BL/6J mice of 4–6 weeks age, weighing 20–25 g were immunized with Mycobacterium bovis (BCG) by the aerogenic route to establish around 1,000–2,000 bacilli of BCG in each mouse (Bhaskar & Upadhyay, 2003). Mice were house in ventilated cages and fed with autoclaved acidified water and irradiated food ad libitum and were kept in 12 h light and 12 h dark conditions.\n\nUse of animals in this investigation was approved by the Institutional animal ethical committee of National Institute of Immunology, New Delhi (IAEC#354/14).\n\nAt every time point of day 1, 7, 14 and 30 post-immunization, two immunized mice were euthanized by an overdose of Ketamine and Xylazine given intraperitoneally. Typically, 35 mg ketamine and 3.5mg xylazine in 350 µl saline per mouse was used to euthanize a mouse.\n\nTheir lung and spleen were isolated aseptically and homogenized in 1 ml PBS using a tissue homogenizer (Polytron PT 1600E, Germany) at 30,000 rpm for 40 s. The homogenized mix was diluted 5 times in PBS and 100 µl diluted mix was spread on the aforementioned agar plates in triplicate.\n\nAt every time point the tissue homogenates were plated on media plates prepared using CAM and commercial BD-Panta antibiotic mixture.\n\nPlates were incubated at 37°C for 4 weeks.\n\nGraphPad Prism 7 software was used to calculate p-values by two-way ANOVA.\n\n\nResults\n\nAfter incubation, BCG colonies present on plates were counted, the results of which are shown in Figure 1. The data (Dataset 1) confirm that on prepared agar plates BCG was able to selectively grow from a complex micro-flora of the lung and spleen. Similar number of BCG colonies were observed on CAM plates and BD-PANTA plates; the differences between the two were statically insignificant (Figure 1).\n\nMycobacterium bovis (BCG) colonies in the lung (A) and spleen (B) after immunization. At each indicated time point, the lung and spleen were isolated from immunized mice followed by single-cell suspension preparation and plating on 7H11 agar plates with Customized Antibiotic Mix (CAM) or BD-PANTA. The number of colonies were counted and compared between the two groups.\n\n\nConclusions\n\nThe cost of above discussed customized antibiotics mix for preparing 5 l of agar media was around 80 INR (around 1.25 USD) which is almost 1/100th of the cost of commercially available antibiotic formulation for the purpose. This antibiotic mix is highly economical, easy to prepare and can significantly reduce the total cost involved in mycobacterium culture.\n\n\nData availability\n\nDataset 1. Number of BCG bacilli colony forming units (CFUs) on each plate from each experimental group. The data show CFUs on each plate along with calculated bacilli load of the tissue. http://dx.doi.org/10.5256/f1000research.14467.d201182 (Kesarwani et al., 2018).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by a grant received from Department of Science and Technology, Government of India (SB/SO/HS/204/2013) and the core grant received from the Department of Biotechnology, Government of India to National Institute of Immunology, New Delhi.\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\nBhaskar S, Upadhyay P: Design and evaluation of an aerosol infection chamber for small animals. Int J Pharm. 2003; 255(1–2): 43–48. PubMed Abstract \| Publisher Full Text\n\nBrogden RN, Carmine AA, Heel RC, et al.: Trimethoprim: a review of its antibacterial activity, pharmacokinetics and therapeutic use in urinary tract infections. Drugs. 1982; 23(6): 405–430. PubMed Abstract \| Publisher Full Text\n\nCardoso LS, Araujo MI, Góes AM, et al.: Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis. Microb Cell Fact. 2007; 6: 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDonovick R, Gold W, Pagano JF, et al.: Amphotericins A and B, antifungal antibiotics produced by a streptomycete. I. In vitro studies. Antibiot Annu. 1955; 3: 579–586. PubMed Abstract\n\nKesarwani A, Nagpal P, Mishra A, et al.: Dataset 1 in: An inexpensive and easy-to-make customized antibiotics mix for mycobacterium culture. F1000Research. 2018. Data Source\n\nLambrecht RS, Carriere JF, Collins MT: A model for analyzing growth kinetics of a slowly growing Mycobacterium sp. Appl Environ Microbiol. 1988; 54(4): 910–916. PubMed Abstract \| Free Full Text\n\nMesa-Arango AC, Scorzoni L, Zaragoza O: It only takes one to do many jobs: Amphotericin B as antifungal and immunomodulatory drug. Front Microbiol. 2012; 3: 286. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO’Keeffe J, Doyle S, Kavanagh K: Exposure of the yeast Candida albicans to the anti-neoplastic agent adriamycin increases the tolerance to amphotericin B. J Pharm Pharmacol. 2003; 55(12): 1629–1633. PubMed Abstract \| Publisher Full Text\n\nPommier Y, Leo E, Zhang H, et al.: DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem Biol. 2010; 17(5): 421–433. PubMed Abstract \| Publisher Full Text\n\nSanders CC: Azlocillin: a new broad spectrum penicillin. J Antimicrob Chemother. 1983; 11(Suppl B): 21–31. PubMed Abstract \| Publisher Full Text" }	[ { "id": "33548", "date": "09 May 2018", "name": "Amit 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\nThis is a very simple paper based on common microbiological techniques. Authors have taken a few common antibiotics and made a cocktail which is cost-effective and gave results comparable to the existing commercially available antibiotics (PANTA). Experiments are planned well, appropriately described, and conclusions are justified. The comparable findings obtained using this highly cost-effective formulation relative to commercially available formulations can be of great help to researchers in TB field.\n\nTo check the reliability of the new formulation, authors have conducted animal experiments and enumerated bacillary load in the infected organs such as lungs and spleen. Authors detected no significant difference between commercial and the new mixture of antibiotics on the growth potential of the BCG strains isolated from the infected lungs. The statistical analysis was done using two-tailed ANOVA, which is most appropriate.\n\nWhile experiments are reliable and results are presented well, I would have liked a control wherein authors plate organ homogenates on the culture media plates without any antibiotic mix. This would have confirmed that the normal microflora present in these organs are interfering with the outgrowth of BCG cells under the experimental conditions used by the authors. This way authors can bolster the efficacy of the antibiotic combination in reducing contamination in the mycobacterial cultures.\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": [] }, { "id": "34517", "date": "04 Jun 2018", "name": "Bhupendra 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 authors have addressed a very pertinent issue of growing mycobacterial culture using a customized cocktail of commonly available antibiotics instead of commercially available expensive antibiotics (PANTA), presently in use. They have performed whole experiments in a very simple way and succinctly described the methods with appropriate conclusions. Since the findings obtained using CAM were comparable to the commercially available PANTA, this can be really useful to researchers in TB field.\n\nBypassing the routine culture and BACTEC methods authors tested CAM in animal based study, which gave ample scope to new mixture of antibiotics to eradicate any prevailing contamination carried forwarded through organ homogenate. However, a control carrying only organ homogenates on the culture media plates without any antibiotic mix would have given fair idea of prevailing microbial contamination and allowed authors to assess how effectively CAM exterminated the microbial flora under the experimental conditions used by authors.\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": [] } ]	1	https://f1000research.com/articles/7-507
https://f1000research.com/articles/7-502/v1	26 Apr 18	{ "type": "Research Note", "title": "Strong evidence that callous–unemotional traits are not related to risk-taking task performance", "authors": [ "Luna C. M. Centifanti", "James Negen", "James Negen" ], "abstract": "A hypothesized association between callous–unemotional (CU) traits and risk-taking may account for the link between CU traits and real-world risky behaviors, such as illegal behavior. Prior findings show that reward and punishment responsivity differs in relation to CU traits, but is not associated with general risk-taking. However this has only been examined previously with one task, only with a frequentist framework, and with limited interpretation. Here, we expand to another task and to Bayesian analyses. A total of 657 participants (52% female) completed the Inventory of Callous–Unemotional Traits, the Balloon Analogue Risk Task (essentially a gambling task), and the Stoplight driving task, which repeatedly presents participants with riskier or less risky choices to make while driving. We found strong evidence for the null model, in which there is no relation between the two risk-taking tasks and CU traits (R2 = 0.001; BF10 = 1/60.22). These results suggest that general risk-taking does not underlie the real-world risky behavior of people with CU traits. Alternative explanations include a different method of valuing certain outcomes.", "keywords": [ "Callous–unemotional traits", "risk taking", "decision making", "driving", "gambling", "Bayesian", "re-analysis" ], "content": "Introduction\n\nCallous–unemotional (CU) traits are an aspect of psychopathy, which includes traits such as callously using others for one’s personal gain, a lack of caring for society’s values and lacking emotional depth1. Risk-taking includes choosing behaviors with uncertain outcomes (but possibly higher rewards) over behavior with more certainty in its rewards2. Here, we show that the two are unrelated when measured in a laboratory setting.\n\nThis is surprising for three reasons. First, a variety of risky real-world behaviors and illegal behaviors1—themselves risky—are associated with CU traits (e.g., substance use, sexual risk-taking)3–7. Second, there is a difference in reward and punishment responsivity in relation to CU traits4,6,8–12. For example, in a test of gambling, the Balloon Analogue Risk Task (BART), CU was related to weaker reward responsivity, in that adolescents with these traits failed to show an increase in risk-taking following successful (rewarded) trials4,13. Third, CU traits are one aspect of a cluster of traits known as psychopathy, which is associated with risk-taking14–16.\n\nThese data were originally collected as part of a study about the influence of peer presence on risk-taking behavior, with two laboratory tasks conducted13,17. CU traits were measured as a potential moderator. Results on the relationship between CU traits and a gambling task have been previously reported using frequentist methods, but the null finding failed to be interpreted13. Here, we re-analyze the data in a Bayesian framework, allowing for the relationship between CU traits and gambling to be interpreted. In addition, for the first time, we report our findings on the association between CU traits and a driving risk-taking task17.\n\n\nMethods\n\nA total of 675 people (52% female; 16–18 years of age) from six schools in Northwest England participated in 2010. Heads of schools acted in loco parentis, and verbal consent was obtained to ensure privacy, which was approved by the ethics committee, within the schools where the research was conducted. Ethical approval was given by the University of Central Lancashire to the first author (PSY0809122). Complete information about the sample and recruitment can be found in a previous report17.\n\nA total of 657 participants produced usable data on all three measures reported here. The Inventory of Callous Unemotional Traits (ICU)18–20, a self-reporting questionnaire, was used to assess CU traits. The BART21, where participants can repeatedly gamble by pumping a balloon for greater reward but risk popping it and receiving no reward, was one measure of risk-taking. The Stoplight driving task22, where participants repeatedly choose to either enter yellow/red lights and risk time-consuming crashes or stop and then proceed on the green light, was also given in counterbalanced order as an additional risk-taking task. All three are standard choices that have been validated19,21,23. At the time of writing, the ICU and BART tasks can be obtained online, and the Stoplight can be obtained by contacting the authors22. Unrelated to the aims of the present study, participants were asked to bring two friends of the same gender and completed the tasks either in their presence or not.\n\nMultiLevel Data Manipulations were conducted in MLwiN 2.30 (University of Bristol, 2014), resulting in an outcome variable for each task that was adjusted to be equated across peer group membership. Descriptive statistics, zero-order Perason correlations and p-values were calculated using JASP 0.8.2.024. An online tool was used to calculate Bayes factors25.\n\n\nResults\n\nFigure 1 shows scatterplots of the relations among the three variables. There was a significant zero-order correlation between the tasks, r=0.22, p<0.001, but not between the ICU scores and either the BART, r=0.033, p=0.397, N=657, BF10=1/8.09 or the driving task, r=0.013, p=0.738, N=672, BF10=1/11.00. More importantly, a multiple linear regression, with the risk-taking tasks predicting ICU scores, showed no significant relation to the BART, β=0.033, t=0.824, p=0.410, or the driving task, β= -0.000, t= -0.012, p=0.990. The overall fit was F (2, 654)=0.359, p=0.698; R2=0.001, R=0.033, N=657, BF10=1/60.22. In a Bayesian analysis, this is considered strong evidence for the null hypothesis25,26.\n\nScores were adjusted for peer-level clustering, since participants were recruited with two friends.\n\nWe also examined the comparability of our sample to others. The mean ± SD for total ICU (21.62 ± 7.85) was comparable to previous community and at-risk samples. For example, our scores were similar to those from a community sample (male, 25.25 ± 7.90; female, 21.76 ± 9.4)27, as well as to youths from a residential facility (25.74 ± 7.95)28.\n\n\nDiscussion\n\nThe results of this study rule out a specific theory about why CU traits are related to risky real-world behaviors including illegal behavior. People with CU traits are not more likely to engage in risky behavior in a lab setting, so real-world risky behaviors are unlikely to be driven by risk-seeking for its own sake. More broadly, this is a worked demonstration that differences in reward and punishment responsivity on a task do not necessarily imply differences in overall risk-taking, even in the same dataset. On the basis of previously reported findings13,17 and our re-analyses, we conclude that these two concepts should not be used interchangeably in interpreting risk-taking results.\n\nThere are potential alternative explanations for why people with high CU traits tend to do risky things, like having unprotected sex. For one, they may simply place different values on the outcomes of catching a disease and/or seeking bodily sensations. However, an interaction between CU traits and antisocial behavior (i.e., conduct disorder) has shown effects on laboratory risk-taking23. One broad possibility is that CU traits do not operate singly, since psychopathy is multifaceted, and some factors of psychopathy appear to be more reliably related to risk taking than others14,30.\n\nPeople who engage in antisocial behavior suffer legal, educational and socio-economic consequences31, and we know CU traits predict antisocial behavior32,33. Thus, further research is needed to understand the mechanisms by which people with CU traits (i) engage in antisocial behavior, and (ii) fail to care about the consequences of their behavior on themselves and on other people. The present study sheds light on one part of this, by showing that one obvious idea of how CU traits and illegal behavior relate is not tenable.\n\n\nData availability\n\nDataset 1. Subject demographic information, together with Inventory of Callous–Unemotional Traits score and results of the tasks. Data are provided in raw form and peer-level adjusted format within the same spreadsheet29. Condition: peer present, 1; peer absent, 0; Subject ID, anonymized participant ID number; Female: female gender, 1; male gender, 0; Age, age in years; BART Pumps AdjAvg_raw data, adjusted average pumps; Peer group level adjusted BART Pumps, peer-group level-adjusted adjusted average pumps; Peer ID, peer group membership ID number; Stoplight Intersections_raw data, number of intersections entered on the Stoplight driving task; Peer group level adjusted Stoplight, peer-group level-adjusted number of intersections entered on the Stoplight driving task; Total ICU, number of CU traits using the Inventory of Callous-Unemotional Traits.\n\nDOI: 10.5256/f1000research.14623.d20181829", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe research presented here was possible with funding from the British Academy, project SG100982, given to the first author. No other funding sources were declared.\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 wish to acknowledge the help of Dr Susanne MacLellan, who assisted with data collection. Thanks, also, to Helen Gowling and Jo McBoyle, who assisted with coding and data entry.\n\n\nReferences\n\nFrick PJ, Ray JV, Thornton LC, et al.: Annual research review: A developmental psychopathology approach to understanding callous-unemotional traits in children and adolescents with serious conduct problems. J Child Psychol Psychiatry. 2014; 55(6): 532–548. PubMed Abstract \| Publisher Full Text\n\nMills B, Reyna VF, Estrada S: Explaining contradictory relations between risk perception and risk taking. Psychol Sci. 2008; 19(5): 429–433. PubMed Abstract \| Publisher Full Text\n\nBaskin-Sommers AR, Waller R, Fish AM, et al.: Callous-Unemotional Traits Trajectories Interact with Earlier Conduct Problems and Executive Control to Predict Violence and Substance Use Among High Risk Male Adolescents. J Abnorm Child Psychol. 2015; 43(8): 1529–1541. PubMed Abstract \| Publisher Full Text\n\nMarini VA, Stickle TR: Evidence for deficits in reward responsivity in antisocial youth with callous-unemotional traits. Personal Disord. 2010; 1(4): 218–229. PubMed Abstract \| Publisher Full Text\n\nRay JV, Thornton LC, Frick PJ, et al.: Impulse Control and Callous-Unemotional Traits Distinguish Patterns of Delinquency and Substance Use in Justice Involved Adolescents: Examining the Moderating Role of Neighborhood Context. J Abnorm Child Psychol. 2016; 44(3): 599–611. PubMed Abstract \| Publisher Full Text\n\nWymbs BT, McCarty CA, King KM, et al.: Callous-unemotional traits as unique prospective risk factors for substance use in early adolescent boys and girls. J Abnorm Child Psychol. 2012; 40(7): 1099–110. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThornton LC, Frick PJ, Ray JV, et al.: Risky Sex, Drugs, Sensation Seeking, and Callous Unemotional Traits in Justice-Involved Male Adolescents. J Clin Child Adolesc Psychol. 2017; 1–12. PubMed Abstract \| Publisher Full Text\n\nBlair RJ: The amygdala and ventromedial prefrontal cortex: functional contributions and dysfunction in psychopathy. Philos Trans R Soc Lond B Biol Sci. 2008; 363(1503): 2557–2565. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlair RJ, Colledge E, Murray L, et al.: A selective impairment in the processing of sad and fearful expressions in children with psychopathic tendencies. J Abnorm Child Psychol. 2001; 29(6): 491–498. PubMed Abstract \| Publisher Full Text\n\nMitchell DG, Colledge E, Leonard A, et al.: Risky decisions and response reversal: Is there evidence of orbitofrontal cortex dysfunction in psychopathic individuals? Neuropsychologia. 2002; 40(12): 2013–2022. PubMed Abstract \| Publisher Full Text\n\nPardini DA, Byrd AL: Perceptions of aggressive conflicts and others’ distress in children with callous-unemotional traits: ‘I’ll show you who’s boss, even if you suffer and I get in trouble’. J Child Psychol Psychiatry. 2012; 53(3): 283–291. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoose A, Bijttebier P, Claes L, et al.: Psychopathic traits in adolescence: Associations with the revised Reinforcement Sensitivity Theory systems. Pers Individ Dif. 2011; 50(2): 201–205. Publisher Full Text\n\nCentifanti LC, Modecki K: Throwing caution to the wind: callous-unemotional traits and risk taking in adolescents. J Clin Child Adolesc Psychol. 2013; 42(1): 106–119. PubMed Abstract \| Publisher Full Text\n\nHosker-Field AM, Molnar DS, Book AS: Psychopathy and risk taking: Examining the role of risk perception. Pers Individ Dif. 2016; 91: 123–132. Publisher Full Text\n\nKastner RM, Sellbom M: Hypersexuality in college students: The role of psychopathy. Pers Individ Dif. 2012; 53(5): 644–649. Publisher Full Text\n\nKhan R, Brewer G, Kim S, et al.: Students, sex, and psychopathy: Borderline and psychopathy personality traits are differently related to women and men’s use of sexual coercion, partner poaching, and promiscuity. Pers Individ Dif. 2017; 107: 72–77. Publisher Full Text\n\nCentifanti LCM, Modecki KL, MacLellan S, et al.: Driving Under the Influence of Risky Peers: An Experimental Study of Adolescent Risk Taking. J Res Adolesc. 2014. Publisher Full Text\n\nEssau CA, Anastassiou-Hadjicharalambous X, Muñoz LC: Psychometric properties of the Spence Children’s Anxiety Scale (SCAS) in Cypriot children and adolescents. Child Psychiatry Hum Dev. 2011; 42(5): 557–568. PubMed Abstract \| Publisher Full Text\n\nKimonis ER, Frick PJ, Skeem JL, et al.: Assessing callous-unemotional traits in adolescent offenders: validation of the Inventory of Callous-Unemotional Traits. Int J Law Psychiatry. 2008; 31(3): 241–252. PubMed Abstract \| Publisher Full Text\n\nRoose A, Bijttebier P, Decoene S, et al.: Assessing the affective features of psychopathy in adolescence: a further validation of the inventory of callous and unemotional traits. Assessment. 2010; 17(1): 44–57. PubMed Abstract \| Publisher Full Text\n\nLejuez CW, Read JP, Kahler CW, et al.: Evaluation of a behavioral measure of risk taking: The Balloon Analogue Risk Task (BART). J Exp Psychol Appl. 2002; 8(2): 75–84. PubMed Abstract \| Publisher Full Text\n\nChein J, Albert D, O’Brien L, et al.: Peers increase adolescent risk taking by enhancing activity in the brain’s reward circuitry. Dev Sci. 2011; 14(2): F1–F10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFanti KA, Kimonis ER, Hadjicharalambous MZ, et al.: Do neurocognitive deficits in decision making differentiate conduct disorder subtypes? Eur Child Adolesc Psychiatry. 2016; 25(9): 989–996. PubMed Abstract \| Publisher Full Text\n\nJASP Team: JASP (Version 0.8.2). 2018.\n\nRouder JN, Morey RD: Default Bayes Factors for Model Selection in Regression. Multivariate Behav Res. 2012; 47(6): 877–903. PubMed Abstract \| Publisher Full Text\n\nKass RE, Raftery AE: Bayes Factors. J Am Stat Assoc. 1995; 90(430): 773–795. Publisher Full Text\n\nMuñoz LC, Qualter P, Padgett G: Empathy and bullying: Exploring the influence of callous-unemotional traits. Child Psychiatry Hum Dev. 2011; 42(2): 183–196. PubMed Abstract \| Publisher Full Text\n\nLui JH, Barry CT, Sacco DF: Callous-unemotional traits and empathy deficits: Mediating effects of affective perspective-taking and facial emotion recognition. Cogn Emot. 2016; 30(6): 1049–1062. PubMed Abstract \| Publisher Full Text\n\nCentifanti LCM, Negen J: Dataset 1 in: Strong evidence that callous–unemotional traits are not related to risk-taking task performance. F1000Research. 2018. Data Source\n\nSwogger MT, Walsh Z, Lejuez CW, et al.: Psychopathy and Risk Taking among Jailed Inmates. Crim Justice Behav. 2010; 37(4): 439–452. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScott S, Knapp M, Henderson J, et al.: Financial cost of social exclusion: follow up study of antisocial children into adulthood. BMJ. 2001; 323(7306): 191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcMahon RJ, Witkiewitz K, Kotler JS, et al.: Predictive validity of callous-unemotional traits measured in early adolescence with respect to multiple antisocial outcomes. J Abnorm Psychol. 2010; 119(4): 752–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuñoz LC, Frick PJ: The reliability, stability, and predictive utility of the self-report version of the Antisocial Process Screening Device. Scand J Psychol. 2007; 48(4): 299–312. PubMed Abstract \| Publisher Full Text" }	[ { "id": "33540", "date": "08 May 2018", "name": "Carlo Garofalo", "expertise": [ "Reviewer Expertise Clinical forensic psychology" ], "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\nThanks for the opportunity to review this manuscript. The study described has important strengths, such as the large sample, the use of well-validated assessment methods, and the sophisticated statistical techniques. I also extremely appreciate the fact that full data are made available, something that happens too rarely to the detriment of the field. The topic is certainly an important one, and one that historically attracts speculations that permeates popular beliefs on the nature and dynamics of callous-unemotional traits. I therefore think that the publication of (null) findings like this is imperative as to not fuel misconceptions on the topic.\nWhile I am therefore clearly positive regarding this submission, I also list below some issues that occurred to me while reading the manuscript, which I think may deserve further elaboration to strengthen the contribution of this study to the literature. I suspect that the writing was constrained by length limits, which could make it impossible to address my suggestions thoroughly, but I thought I would nevertheless mention what I believe could further improve this well-executed and well-written study.\nIt is clear to me that the findings do not support the hypothesis that CU traits are related to risk taking. But I could not tell: who proposed this link, and whether this hypothesis also guided the present research. I think it would help situate the present study within the broader literature on CU if the manuscript could (briefly) refer whether the CU-risk taking link was just one that ‘makes sense’, or one that is central in existing theories of CU (e.g., in the opening of the discussion, it is stated that the present study rules out a ‘specific theory’, but it is unclear which theory is referred to). I think this is important because such theories would have to be re-considered in light of this null finding (as opposed to the scenario in which the CU-risk taking link is more of a popular misconceptions with no footing in scholarly work). Relatedly, as I find the alternative explanation (i.e., ‘a different method of valuing certain outcomes’) equally – if not more – compelling, I think it would be helpful to know whether this possibility was already acknowledged before the study was conducted, or followed the null findings that did not support the initial hypothesis.\n\nA second conceptual clarification that may be needed concern the overlap and dissociation between reward/punishment sensitivity and risk-taking. This seems relevant to understand specific abnormalities related to CU traits and what they can mean. Related to this – it looks like CU traits were also not related in punishment sensitivity, if I am not misunderstanding. If that is the case, how does this null finding aligns with existing theories of psychopathy such as the low-fear hypothesis or the response modulation model?\n\nFor non-familiar readers (as myself), it may be worth expanding on how the Bayesian framework adopted allows for an interpretation of the null finding in a way that frequentist methods would not (also just a brief mention of why the overall fit can be considered strong evidence of null hypothesis).\n\nI cannot help but wonder what the results look like if the ICU is examined at a sub-scale level. I do not expect differences, but I think it is important to rule out that the overall null effect is not due to differential associations between ICU subscales and risk-taking.\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": "33543", "date": "11 May 2018", "name": "Timothy Stickle", "expertise": [ "Reviewer Expertise Callous and unemotional traits", "youth psychopathy", "adolescent substance use", "statistics" ], "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 and is likely to contribute to the literature. There are, however, several areas that need elaboration or clarification to better communicate the findings and their contribution.\n\nUse of Bayesian analysis in a reasonably large sample adds strength to the authors interpretation of null findings.\n\nClarification about some detail of the Bayesian analysis for the reader would be helpful and should include:\n\nHow was the prior selected and what was used for the prior distribution?\n\nWas it a noninformative or an informative prior?\n\nWas sensitivity analysis conducted to gauge the effect of the prior?\n\nIt is worth noting that the sample is reasonably large, so the effect of the prior on at least some parameters may be small. Nevertheless, it would be helpful for the authors to note the effect of sample size on prior and posterior probabilities.\n\nIn the first paragraph of the Discussion section, the authors make a strong declarative statement that the results “…rule out a specific theory about why CU traits are related to risky real-world behaviors... People with CU traits are not more likely to engage in risky behavior in a lab setting, so real-world risky behaviors are unlikely to be driven by risk-seeking for its own sake.”\n\nAlthough the following statements regarding caution in generalizing reward and punishment processing in laboratory tasks to behavior outside the lab clearly are warranted, the above statements seem a step too far. That is, the results clearly cast some doubt on the links and mechanisms of risk. The notion that the finding is conclusive, however, does not seem warranted. Rather, it is recommended that the authors use language to indicate that the current results fail to support links between the specific measure of risk used here and CU traits. It is a reasonable inference to note that the presence of CU traits alone and documented mechanisms of reward and punishment processing are not de facto predictors of risk. It seems more defensible to state something more akin to noting that the currents results call for reexamination of the mechanisms thought to link CU traits and risky behaviors.\n\nIn extending these recommended comments, it would also be helpful for the authors to synthesize the discussion paragraphs a bit more in their discussion of the multifaceted/multidimensional nature of psychopathy and the implications of the broader psychopathy construct in understanding risk and its mechanisms.\n\nOverall, the paper is interesting and the above comments are offered in the spirit of strengthening how the results are communicated.\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? 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": [] } ]	1	https://f1000research.com/articles/7-502
https://f1000research.com/articles/7-499/v1	26 Apr 18	{ "type": "Case Report", "title": "Case Report: Exome sequencing reveals recurrent RETSAT mutations and a loss-of-function POLDIP2 mutation in a rare undifferentiated tongue sarcoma", "authors": [ "Jason Y. K. Chan", "Peony Hiu Yan Poon", "Yong Zhang", "Cherrie W. K. Ng", "Wen Ying Piao", "Meng Ma", "Kevin Y. Yip", "Amy B. W. Chan", "Vivian Wai Yan Lui", "Peony Hiu Yan Poon", "Yong Zhang", "Cherrie W. K. Ng", "Wen Ying Piao", "Meng Ma", "Kevin Y. Yip", "Amy B. W. Chan" ], "abstract": "Soft tissue sarcoma of the tongue represents a very rare head and neck cancer with connective tissue features, and the genetics underlying this rare cancer are largely unknown. There are less than 20 cases reported in the literature thus far. Here, we reported the first whole-exome characterization (>×200 depth) of an undifferentiated sarcoma of the tongue in a 31-year-old male. Even with a very good sequencing depth, only 19 nonsynonymous mutations were found, indicating a relatively low mutation rate of this rare cancer (lower than that of human papillomavirus (HPV)-positive head and neck cancer). Yet, among the few genes that are somatically mutated in this HPV-negative undifferentiated tongue sarcoma, a noticeable deleterious frameshift mutation (with a very high allele frequency of >93%) of a gene for DNA replication and repair, namely POLDIP2 (DNA polymerase delta interacting protein 2), and two recurrent mutations of the adipogenesis and adipocyte differentiation gene RETSAT (retinol saturase), were identified. Thus, somatic events likely affecting adipogenesis and differentiation, as well as potential stem mutations to POLDIP2, may be implicated in the formation of this rare cancer. This identified somatic whole-exome sequencing profile appears to be distinct from that of other reported adult sarcomas from The Cancer Genome Atlas, suggesting a potential unique genetic profile for this rare sarcoma of the tongue. Interestingly, this low somatic mutation rate is unexpectedly found to be accompanied by multiple tumor protein p53 and NOTCH1 germline mutations of the patient’s blood DNA. This may explain the very early age of onset of head and neck cancer, with likely hereditary predisposition. Our findings are, to our knowledge, the first to reveal a unique genetic profile of this very rare undifferentiated sarcoma of the tongue.", "keywords": [ "Head and neck", "Sarcoma of the tongue", "low mutational burden", "POLDIP2 and RETSAT mutations" ], "content": "Introduction\n\nSoft tissue sarcomas of the head and neck are an uncommon heterogeneous group of malignancies1, with the most common subtype being malignant fibrous histiocytoma2,3. Yet, undifferentiated sarcoma of the tongue represents an even rarer tumor within this heterogeneous group of sarcomas with limited treatment options. Thus far in the literature, only about 20 cases have been reported4. At the time of writing, the underlying genetic aberrations of this rare undifferentiated soft tissue cancer of the tongue remains unknown. Here, we describe a case report of an undifferentiated sarcoma of the tongue with associated pathological analysis and whole-exome sequencing (WES) of the tumor to identify the underlying genetic changes and attempt to determine if there exists any potential druggable mutations for treatment.\n\n\nCase report\n\nA 31-year-old Chinese male who had a 1-pack-year smoking history, was a non-drinker, had no significant past medical history, no exposure to radiation, no family history of carcinomas, particularly no family history of members with young onset malignancies, presented in December of 2016 with an anterior tongue mass. Patient biopsy revealed it to be an undifferentiated sarcoma. Further magnetic resonance imaging (MRI) demonstrated a lesion localized to the tongue that involved the extrinsic tongue musculature. Subsequently the patient underwent a total glossectomy with bilateral selective neck dissection levels I–III with an anterolateral thigh-free-flap reconstruction in January 2017. The pathological findings from the surgical specimen are further described below. WES analyses were performed on the fresh surgically resected tumor (tumor content, >50%) and a paired blood sample. Adjuvant chemotherapy and radiation were recommended for this patient, but both options were declined because of concerns regarding long term toxicity and the effects on speech and swallowing. As of October 2017, the patient was disease-free with no documented recurrences on repeated MRI of the oral cavity and neck.\n\nThe tumor mass measured grossly 7 × 7 × 5.5 cm on examination following resection. On microscopic examination, the tumor was composed of mostly spindle cells arranged in short fascicles or a vague storiform pattern. Frequent mitotic figures and prominent tumoral necrosis were seen. No osseous, chondroid or rhadomyomatous differentiation was seen on haematoxylin and eosin staining (Figure 1–Figure 3). The closest margins were 2 mm and located at the postero-inferior, right and left margins.\n\nThe surface squamous epithelium shows no dysplastic field change or connection with sarcoma. (H&E stain; magnification, ×20).\n\n(H&E stain; magnification, ×40).\n\nMitotic figures were frequently noted. No osseous, chondroid or rhadomyomatous differentiation was seen. (H&E stain; magnification, ×400).\n\nFurther immunohistochemical studies showed that the tumor cells were positive for vimentin and p16; had focal weak positivity for SMA; had moderate positivity for TLE-1; and were negative for p63, EMA, AE1/AE3 antibodies, c-Kit, S100, CD31, CD34, desmin, myogenin, melan A, HMB45 antibody, ALK-1, human herpesvirus 8 (HHV8), CD99 (MIC-2) and SRY-box 10 (SOX10). Calponin was equivocal. No loss of integrase interactor 1 (INI1) staining was noted. PCR for high-risk human papillomavirus (HPV) DNA was negative. The Molecular Break-Apart FISH test for SS18 translocation was negative. Overall the pathological features demonstrated a high-grade sarcoma with no definite line of differentiation, consistent with an undifferentiated sarcoma.\n\nWES at >200x depth (Illumina HiSeq 4000) was performed to determine the genetic aberrations underlying this rare form of head and neck cancer. Normal DNA from the patient’s blood was also subjected to WES at the same depth. Surprisingly, WES revealed only 19 non-synonymous mutations in the tumor. The very low rate of non-synonymous mutations is uncommon in HPV-negative and HPV-positive head and neck squamous cell carcinoma (HNSCC)5, and is slightly lower than that of reported sarcomas.\n\nThe somatically mutated genes were, in the order of allele frequencies of these 19 mutational events, POLDIP2 (DNA polymerase delta interacting protein 2), tubulin gamma complex associated protein 3TUBGCP3,, mutated in colorectal cancers (MCC), TUBA3D (tubulin alpha 3d), DDX11 (DEAD/H-box helicase 11), CWF19L2 (CWF19 like 2, cell cycle control), ZNF91 (zinc finger protein 91), RETSAT (retinol saturase) (2 mutations), PRR21 (proline rich 21), TAS2R46 (taste 2 receptor member 46), FAM186A (family with sequence similarity 186 member A), HEATR5A (HEAT repeat containing 5A), VPS4B (vacuolar protein sorting 4 homolog B), PRAMEF12 (PRAME family member 12), FAM170B (family with sequence similarity 170 member B), BBS4 (Bardet-Biedl syndrome 4), ARHGAP5 (Rho GTPase activating protein 5) and ATAD3B (ATPase family, AAA domain containing 3B) (Table 1). Strikingly, based on the functional annotation of these mutated genes, five of the top seven mutated genes with high allele frequencies (>10%) were known to be involved in DNA replication, and mitosis (Table 1). This suggests that major somatic mutations of this rare tumor appear to affect DNA replication and mitosis, consistent with an aggressive phenotype. Of note, the patient’s tumor carried a 93% allele frequency of the POLDIP2 S28fs mutation, which is a hotspot mutation in multiple cancers. In addition, the MCC gene was also mutated in this rare tumor (MCC p.G20S) at a high allele frequency of 28%, indicating its likely role as a driver event for tumorigenesis. Interestingly, the PRR21 mutation is also found to be mutated at high frequencies in TCGA HNSCC cohort with a hotspot mutation S86Gfs291 and M48Tfs329 mutation, while in this tumor, PRR21 mutation occurred at G114C, a position near to these two hotspot mutations in HNSCC.\n\nMost strikingly, among all the somatic mutations identified in this patient, we found that recurrent somatic events of RETSAT (RETSAT p.A533V and p.G536R mutations), a gene known to be important for the promotion of adipogenesis and normal adipocyte differentiation. It is plausible that RETSAT mutations may affect adipocyte adipogenesis and differentiation, related to the sarcoma features of this rare tumor.\n\nGene copy number analysis was also performed and a total of 221 somatic copy number alterations (CNA) events were identified (Figure 4 and Supplementary Table 1). The somatic CNA detected in this patient did not harbor any of these common HNSCC CNA events including losses of chr. 3p and 8p, as well as focal amplification or gains of chr. 3q26/28, 5p15, and 8q245. The common CNA events in sarcomas including aberrations of the MDM2-p53 and p16-cyclin dependent kinase 4 (CDK4)-retinoblastoma-associated protein pathways, deletion of TP53 (tumor protein p53), CDKN2A (cyclin dependent kinase inhibitor 2A), as well as chr. 12q13-q15 and CDK4 amplification were also absent in this patient6. These results suggest that this unique tumor is likely distinct from HNSCC and adult soft tissue sarcomas.\n\nAn ideogram of a normal karyotype is shown in the outermost ring. Chromosomes are segmented into contiguous bins of 1 Mb in size. The second ring from outside shows CNVs at corresponding chromosomal positions. Distinct heights of red dots indicate different inferred copy numbers. Regions that are not covered by any dots have a copy number of two. The third ring shows SNV density. Purple dots with different heights indicate SNV densities of the corresponding regions. The innermost ring shows positions of genes with non-synonymous mutations found in the undifferentiated sarcoma. CNV, copy number variation; SNV, single nucleotide variant.\n\nGiven the young age of this patient, we also carefully examined potential aberrations in his germline mutational profile by WES of his blood DNA. Importantly, we found that this patient carried multiple germline mutational events of TP53, including a missense mutation of TP53 p.P72R (100% allele frequency), 2 mutations at the immediate 5’-UTR, likely in the promoter region of TP53 (c.-112G>A; and c.-123C>G; both with 100% allele frequencies), as well as two other intron variants with unknown effects (Table 2). The TP53 germline events likely explain the early age of onset for cancer for this patient. In addition, a 5’-UTR mutation, likely at the promoter region of CDKN2A, was identified (c.*193G>C), with an allele frequency of 100%. A CASP8 (caspase 8) missense mutation, CASP8 p.L14R, was also found in this patient’s blood with a 46.7% allele frequency. However, the potential function of this mutation is unclear. Lastly, this patient carries as many as 23 germline variants of NOTCH1; however, none of these variants are non-synonymous mutations, and the effects of these variants are largely unknown. No telomerase reverse transcriptase promoter or exon mutation was found. Lastly, no germline gene copy number alterations were observed for any critical HNSCC-associated oncogenes or suppressor genes (Supplementary Table 2)\n\n\nDiscussion\n\nThe development of soft tissue sarcomas in the oral cavity and head and neck region in general are rare, with the development of an undifferentiated sarcoma in the oral cavity of an even lower likelihood7,8. A US population database analysis of head and neck sarcoma patients identified that the median age of adults affected was 55–59 years old, most commonly affecting the skin and soft tissues2. This contrasts with the young age of our patient at presentation (31 years). The presence of a rare soft tissue sarcoma in a young patient provided the evidence to evaluate for germline mutations in addition to the somatic mutations, leading to the discovery of multiple germline TP53 mutations, as well as multiple CDKN2A, and potentially multiple NOTCH1 mutations of unclear functions. Notably, a CASP8 mutation was also found in his germline. It is important to note that all these genes are known to be somatically mutated in HNSCC5,9.\n\nInterestingly, the somatic mutational profile of this tumor was rather distinct from both HNSCC and sarcomas, with a relatively low number of non-synonymous mutations. Furthermore, unlike the genetic profile of soft tissue sarcomas, our sarcoma patient lacked the common somatic mutations in TP53, PTEN (phosphatase and tensin homolog) and CDKN2A6,10. Moreover, his tumor also lacked the most common somatic mutations of HNSCC, such as TP53, NOTCH1, CKDN2A, PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) and FAT1 (FAT atypical cadherin 1)5,11,12. This was partly contributed to by his heavy germline mutations of some of these key tumor suppressor genes, including TP53 and CDKN2A, which are commonly mutated in both sarcoma and HNSCC, usually somatically. Furthermore, his germline events also carried CASP8 and NOTCH1 mutations, which are commonly somatically mutated in HNSCC rather than in the germline. This difference in mutation profile in conjunction with the young age of the patient led us to consider heavy germline mutations as a possible underlying cause of this rare undifferentiated sarcoma of the tongue.\n\nGermline mutations in TP53 are associated with familial clustering of early onset carcinomas, including pre-menopausal breast cancer, soft tissue sarcomas, adrenal cortical carcinomas or choroid plexus carcinomas, which together are classified as Li–Fraumeni syndrome based on Chompret criteria13–16. Li–Fraumeni-like syndrome represents a subset of patients with familial clustering that do not meet the criteria for Li–Fraumeni syndrome15. However, de novo germline mutations in TP53 have been described, proven and are thought to be relatively common17,18. Our case highlights the importance of considering TP53 mutations in early onset soft tissue sarcomas, even in those without familial histories, as this offers the potential to appropriately manage the patient and possibly the family.\n\nThe development of soft tissue sarcomas in an uncommon location in a young patient indicated the possibility of germline events, despite the lack of a family history of carcinomas at a young age. The early detection of these mutations can be useful in treating these patients, given the consideration that radiation wherever possible should be avoided to prevent the development of radiation-induced second malignancies19–22. Regarding the surveillance for second primary cancers, full-body MRI examinations and positron emission tomography have been recommended, as opposed to exposure to radiation with computed tomography19. This case highlights the need for vigilance and consideration of possible hereditary predisposition syndromes or de novo germline mutations in patients with rare tumours at a young age and with atypical genetic profiles.\n\n\nMethods\n\nTumor tissue and blood sample were collected from the patient under written informed consent according to The Joint Chinese University of Hong Kong – New Territories East Cluster Clinical Research Ethics Committee, Hong Kong SAR (protocol number CRE-2015.396). The tumor mass was freshly frozen for DNA extraction using the QIAGEN DNeasy Blood and Tissue kit. Blood DNA was extracted from patient’s buffy coat in a similar manner. DNA samples were then quantified and quality-assessed using a bioanalyzer.\n\nGenomic DNA of the patient’s tumor and buffy coat were used for WES using the Agilent SureSelect Human All Exon V5 Kit, with sequencing performed using the Illumina HiSeq 4000 platform (Macrogen, Korea) with a goal coverage of ×200 for tumor and ×100 for the blood (buffy coat) sample of the same patient.\n\nUpon sequencing, all reads (FASTQ files) were mapped to the hg19 human reference genome assembly with Burrows-Wheeler Alignment Tool (version 0.7.12). Variant calling of single nucleotide variants (SNVs) and indels was performed using the Genome Analysis Toolkit (version v3.4.0) HaplotypeCaller pipeline. Called variants were annotated with SnpEff (version 4.3), the exome sequencing data have been deposited into European Nucleotide Archive (ENA) with accession number PRJEB25783. Somatic mutations were defined as mutations found in the tumor tissue of the patient but not in the patient’s blood. All identified recurrent mutations were confirmed in Integrative Genomics Viewer (IGV) (version 2.4) as a final check of the calling (100% accurate).\n\nCNAs were analyzed from segmented WES data using the Control-FREEC copy number and genotype caller (version 11.0) in both somatic and germline mode. In both modes, captured genomic regions were characterized using the SureSelect Human All Exon V5 bed file. Somatic CNA analyses were run by referencing tumor WES data to the paired blood WES data, whereas germline CNA analysis were run by referencing blood WES data to the hg19 human genome assembly. Identified regions were annotated with ANNOVAR (version 2017-07-17) and SnpEff. Segments with CNA were manually confirmed with an IGV check. Genetic variations were visualized using Circos (version 0.69) with “chromosomes_unites = 1000000”.\n\nThe patient’s tumor tissue was FFPE-preserved and sectioned (5–8 µm) for immunohistochemical and H&E staining. Immunohistochemical staining was performed with the following antibodies: Melan A (M7196, Dako, Denmark), HMB34 (M0634, Dako, Denmark), ALK-1 (M7195, Dako, Denmark), HHV8 (NCL-HHV8-LNA, Novo, UK), MIC-2 (M3601, Dako, Denmark), SOX-10 (ACI3099, Biocare, USA), Calp (M3556, Dako, Denmark), INI1 (612110, DB transduction, USA), Vimentin (M0725, Dako, Denmark), p16 (805-4713, Ventana, USA), SMA (M0851, Dako, Denmark), p53 (M7001, Dako, Denmark), TLE1 (401M-16, Cell Marque, USA), p63 (M7317, Dako, Denmark), EMA (MS348, Thermo, UK), AE1/3 (M3515, Dako, Denmark), c-kit (A4502, Dako, Denmark), S100 (NCL-S100p, Leica, UK), CD31 (M0823, Dako, Denmark), CD34 (NCL-L-END, Leica, UK), Desmin (M0760, Dako, Denmark) and Myogenin (M3559, Dako, Denmark). Results were examined by an experienced pathologist for the presence or absence of the target proteins. Pictures were taken under a light microscope at ×20, ×40 and ×400 magnification.\n\nThe tumor cells from the formalin-fixed, paraffin-embedded sections were isolated for DNA extraction. The DNA was subjected to PCR analysis using the consensus primers GP5+/6+, as previously documented23.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and accompanying images was obtained from the patient.\n\n\nData availability\n\nThe exome sequencing data have been deposited into European Nucleotide Archive (ENA) with accession number PRJEB25783.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nDr Stanley Ho Medical Foundation (to JYKC); General Research Fund from the Research Grant Council, Hong Kong (#1711484 and #17121616 to VWYL); Theme-based Research from the Research Grant Council, Hong Kong (T12-401/13-R to VWYL); Hong Kong Cancer Fund (to VWYL); and the Direct Grant for Research (#2016.095), the Chinese University of Hong Kong (to VWYL and JYKC).\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. Somatic copy number variations obtained from DNA taken from the patient’s tumor.\n\nClick here to access the data.\n\nSupplementary Table 2. Germline copy number variations obtained from DNA taken from the patient’s blood.\n\nClick here to access the data.\n\n\nReferences\n\nCormier JN, Pollock RE: Soft tissue sarcomas. CA Cancer J Clin. 2004; 54(2): 94–109. PubMed Abstract \| Publisher Full Text\n\nPeng KA, Grogan T, Wang MB: Head and neck sarcomas: analysis of the SEER database. Otolaryngol Head Neck Surg. 2014; 151(4): 627–633. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShuman AG, Brennan MF, Palmer FL, et al.: Soft tissue sarcoma of the head & neck: nomogram validation and analysis of staging systems. J Surg Oncol. 2015; 111(6): 690–695. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNguyen A, Vaudreuil A, Haun P, et al.: Clinical Features and Treatment of Fibrous Histiocytomas of the Tongue: A Systematic Review. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStransky N, Egloff AM, Tward AD, et al.: The mutational landscape of head and neck squamous cell carcinoma. Science. 2011; 333(6046): 1157–1160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi FP, Fraumeni JF Jr, Mulvihill JJ, et al.: A cancer family syndrome in twenty-four kindreds. Cancer Res. 1988; 48(18): 5358–5362. PubMed Abstract\n\nMalkin D, Li FP, Strong LC, et al.: Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science. 1990; 250(4985): 1233–1238. PubMed Abstract \| Publisher Full Text\n\nOlivier M, Hollstein M, Hainaut P: TP53 mutations in human cancers: origins, consequences, and clinical use. Cold Spring Harb Perspect Biol. 2010; 2(1): a001008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonzalez KD, Noltner KA, Buzin CH, et al.: Beyond Li Fraumeni Syndrome: clinical characteristics of families with p53 germline mutations. J Clin Oncol. 2009; 27(8): 1250–1256. PubMed Abstract \| Publisher Full Text\n\nChompret A, Brugières L, Ronsin M, et al.: P53 germline mutations in childhood cancers and cancer risk for carrier individuals. Br J Cancer. 2000; 82(12): 1932–1937. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLalloo F, Varley J, Moran A, et al.: BRCA1, BRCA2 and TP53 mutations in very early-onset breast cancer with associated risks to relatives. Eur J Cancer. 2006; 42(8): 1143–1150. PubMed Abstract \| Publisher Full Text\n\nAgir H, MacKinnon C, Tan ST: Li-Fraumeni syndrome: a case with 4 separate primary sarcomas and 5 sequential free flaps in the maxillofacial region. J Oral Maxillofac Surg. 2008; 66(8): 1714–1719. PubMed Abstract \| Publisher Full Text\n\nVarley J: TP53, hChk2, and the Li-Fraumeni syndrome. Methods Mol Biol. 2003; 222: 117–129. PubMed Abstract \| Publisher Full Text\n\nHisada M, Garber JE, Fung CY, et al.: Multiple primary cancers in families with Li-Fraumeni syndrome. J Natl Cancer Inst. 1998; 90(8): 606–611. PubMed Abstract \| Publisher Full Text\n\nLimacher JM, Frebourg T, Natarajan-Ame S, et al.: Two metachronous tumors in the radiotherapy fields of a patient with Li-Fraumeni syndrome. Int J Cancer. 2001; 96(4): 238–242. PubMed Abstract \| Publisher Full Text\n\nJacobs MV, Snijders PJ, van den Brule AJ, et al.: A general primer GP5+/GP6(+)-mediated PCR-enzyme immunoassay method for rapid detection of 14 high-risk and 6 low-risk human papillomavirus genotypes in cervical scrapings. J Clin Microbiol. 1997; 35(3): 791–795. PubMed Abstract \| Free Full Text" }	[ { "id": "33489", "date": "08 May 2018", "name": "Tsung Lin Yang", "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\nA rare undifferentiated tongue sarcoma showing recurrent RETSAT mutations and a loss-of-function POLDIP2 mutation was presented in this case report. By showing comprehensive genetic analyses, it was interesting to identify the unique presentation of the common genes found in head and neck cancer, including TP53, CDKN2A, and NOTCH1. It is undoubtedly that the genetic profiles of this rare tongue sarcoma is different from those found in the common HNSCC because of distinct pathological background. Nonetheless, the presentations of this case different from the common sarcoma merit further confirmation. If it is really the case, it infers the possibility that the anatomical factor is presumably involved. Data related to the sarcoma of head and neck is suggested to be compared. Although this case might provide a chance to evaluate the mutual influence between the germline and somatic mutation, evidences are required to confirm whether the heavy germline mutation indeed affect the incidence of common somatic mutation found 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? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "33531", "date": "15 May 2018", "name": "Marietta Tan", "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\nUndifferentiated sarcoma of the tongue is a rare malignancy of the head and neck, and its underlying genetic alterations have not previously been investigated. In this case report, the authors performed whole-exome sequencing (WES) of a tongue sarcoma and paired blood sample of a 31-year-old male. They identified a fairly low mutation rate, with mutations in POLDIP2, a gene important in DNA replication and repair, and RETSAT, a gene involved in adipogenesis. WES analysis of the patient’s blood DNA revealed mutations in TP53, CDKN2A, CASP8, and NOTCH1.\nOverall, this is a well-written case report of a rare tumor arising in a patient with an interesting germline mutational profile. The authors should consider the following:\nIs “undifferentiated sarcoma of the tongue” a distinct clinicopathologic entity? The introduction discusses the rarity of undifferentiated sarcomas of the tongue but then references a review of malignant fibrous histiocytoma.\n\nSeveral times throughout the manuscript, the authors compare the mutational profile of the tongue sarcoma to that of head and neck squamous cell carcinoma. They remark that the mutational profiles of the two tumor types are distinct. I would not necessarily expect that the mutational events underlying a mesenchymal-derived tumor would be similar to an epithelial-derived tumor. Can the authors comment on why they expected that the mutational profiles might be similar?\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": "34003", "date": "11 Jul 2018", "name": "Robin Jones", "expertise": [ "Reviewer Expertise Sarcomas and Phase I 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 is an informative and well written report on a very rare cancer, undifferentiated sarcoma of the tongue. The authors could consider discussing the challenges of the pathological classification of sarcomas. For instance, they reference a review by Dr Nguyen and colleagues – this review cites studies dating from the 1980s. There have been clear improvements in the classification of sarcomas over this time. Consequently, this strengthens the importance of the current study which provides detailed exome sequencing data. I agree with other referees that it is not surprising that the profile of an undifferentiated sarcoma is different to that of a head and neck squamous cell carcinoma.\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/7-499
https://f1000research.com/articles/7-340/v1	20 Mar 18	{ "type": "Research Note", "title": "Preliminary investigation of deoxyoligonucleotide binding to ribonuclease A using mass spectrometry: An attempt to develop a lab experience for undergraduates", "authors": [ "Daniel D. Clark" ], "abstract": "Deoxyoligonucleotide binding to bovine pancreatic ribonuclease A (RNase A) was investigated using electrospray ionization ion-trap mass spectrometry (ESI-IT-MS). Deoxyoligonucleotides included CCCCC (dC5) and CCACC (dC2AC2). This work was an attempt to develop a biochemistry lab experience that would introduce undergraduates to the use of mass spectrometry for the analysis of protein-ligand interactions. Titration experiments were performed using a fixed RNase A concentration and variable deoxyoligonucleotide concentrations. Samples at equilibrium were infused directly into the mass spectrometer under native conditions. For each deoxyoligonucleotide, mass spectra showed one-to-one binding stoichiometry, with marked increases in the total ion abundance of ligand-bound RNase A complexes as a function of concentration, but the accurate determination of dC5 and dC2AC2 dissociation constants was problematic.", "keywords": [ "education", "biochemistry lab", "protein-ligand interactions", "mass spectrometry", "ribonuclease A" ], "content": "Abbreviations\n\ndC5 deoxyoligonucleotide with the sequence: CCCCC\n\ndC2AC2 deoxyoligonucleotide with the sequence: CCACC\n\nRNase A bovine pancreatic ribonuclease A\n\nESI-IT-MS electrospray ionization ion-trap mass spectrometry\n\nnESI-Q-TOF-MS nanoelectrospray ionization quadrupole time-of-flight mass spectrometry\n\nRNase A+dC5 ligand-bound form of RNase A (with one dC5 ligand)\n\nRNase A+dC2AC2 ligand-bound form of RNase A (with one dC2AC2 ligand)\n\nRSD relative standard deviation\n\n\nIntroduction\n\nBovine pancreatic ribonuclease A (RNase A) is an endoribonuclease (EC 3.1.27.5) that hydrolyzes RNA. It is a small single chain polypeptide (124 amino acids) containing four disulfide bridges and is known for its significant stability1. RNase A has been called “the most studied enzyme of the 20th century” and it has seen wide use as a model protein in biochemical and biophysical experiments1. Undergraduate life-science majors often learn of RNase A as part of a biochemistry course in the context of the Nobel Prize winning protein folding experiments performed by Christian Anfinsen2. Students may also be familiar with the need to inhibit ribonucleases when working with RNA in the lab, often accomplished with diethyl pyrocarbonate, or will have learned about the role of ribonucleases in microRNA biology3. Still others may recognize RNase A as an example of an enzyme that employs general acid-base catalysis as part of its chemical mechanism4. Thus, RNase A is an excellent model for undergraduate lab experiments, not only because it has been extensively studied, but also because its use presents an opportunity to reemphasize important concepts in biochemistry and biology.\n\nThe application of mass spectrometry to the analysis of biomolecules has made an enormous impact in the life sciences. Protein identification, the characterization of protein modifications, and the quantification of biomolecules using mass spectrometry are commonplace. Of these, protein identification is the most established in an undergraduate teaching lab5–10. Numerous other biological applications of mass spectrometry have existed for many years, but some of these are arguably, less broadly appreciated, and this is especially true for undergraduates. Native mass spectrometry is an approach based on electrospray ionization, where biomolecules are sprayed from a non-denaturing solvent11. Under such conditions, protein-ligand complexes can be maintained and a dissociation constant (Kd) can be determined via a titration experiment12–14.\n\nPreviously, nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nESI-Q-TOF-MS) was used to investigate ligand binding to RNase A12,15,16. These studies used nESI ionization for its superior sensitivity and relied on the TOF mass analyzer for its high mass range12,15,16. In Zhang et al., free RNase A and the ligand-bound forms of RNase A populated three charge states (+8, +7, and +6) at pH 6.6, with most of the signal (~90%) coming from the +7 charge state, which exceeded m/z 2000 in the ligand-bound forms12. Similarity, in Sundqvist et al., focus was placed on the +7 charge state of free RNase A and its ligand-bound forms15. In contrast, Yin et al. reported the most abundant charge state of free and ligand-bound forms of RNase A to be +8 at pH 6.616. Unfortunately, California State University-Chico does not own a nESI-Q-TOF-MS as employed by each of these research groups. Instead, we have an electrospray ionization ion-trap mass spectrometer (ESI-IT-MS), which by comparison to nESI-Q-TOF-MS, offers a lower sensitivity and mass range (50–2000 m/z). Consequently, at the outset of this preliminary investigation, it was recognized that observation of the +7 and +6 charge states of ligand-bound RNase A would not be possible with our instrument.\n\nThis work was an attempt to develop a biochemistry lab experience that would introduce undergraduate life-science majors to the use of mass spectrometry for the analysis of protein-ligand interactions. Two deoxyoligonucleotides, CCCCC (dC5) and CCACC (dC2AC2), were investigated for their ability to bind RNase A. Titration experiments were performed using a fixed RNase A concentration and variable deoxyoligonucleotide concentrations. Samples at equilibrium were infused directly into our ESI-IT-MS under native conditions. The relative simplicity of the sample preparation and instrument operation (by direct infusion) were viewed as desirable features for an undergraduate teaching lab. Data analysis was also straightforward. Herein is described the results of this preliminary investigation. This work differentiates itself from the abovementioned RNase A ligand binding studies (using mass spectrometry) by the experimental conditions employed, which includes the identity of the investigated ligands and the type of mass spectrometer used12,15,16.\n\n\nMethods\n\nA stock solution of bovine pancreatic ribonuclease A (#R6513, Sigma-Aldrich, St Louis, MO, USA) was prepared at 5.60 mg/mL in LC-MS grade water (Thermo-Fisher Scientific, Waltham, MA, USA). Ammonium acetate (NH4OAc) was LC-MS grade (#73594, Sigma-Aldrich). HPLC-purified deoxyoligonucleotides with the sequence “CCCCC” (dC5) and “CCACC” (dC2AC2) were obtained from ThermoFisher and the stock solutions (200 μM) were prepared in LC-MS grade water. Samples were prepared in 1.5 mL microcentrifuge tubes as indicated in Table 1. Six replicates were prepared and analyzed for “Sample 1” whereas “Samples 2–5” were prepared and analyzed in triplicate. Each sample was mixed by micropipetting, and incubated at room temperature for ten minutes, prior to analysis.\n\n1409 μM RNase A; calculated with the MWav (13,690.3) for PDB ID:1RTA (Ref. 17).\n\n2Either dC5 or dC2AC2.\n\nSamples were analyzed with a Thermo LCQ Advantage ion-trap mass spectrometer equipped with an electrospray ionization source. The instrument was operated in positive ion mode using a 4.5 kV spray voltage, 60°C capillary temperature, 200 ms inject time, 10 microscans, and nitrogen sheath and aux gas settings of 30 and 15, respectively. The instrument was tuned on the +8 charge state of free RNase A at m/z 1723.7 (Table 2). Each sample was subjected to direct-infusion at 2.5 µL/min using the LCQ syringe pump and full-scan mass spectra (m/z 1500-1950) were collected for two minutes. The upper m/z range was capped at 1950 to exclude the +7 charge state of free RNase A, which in its various adduct forms, began at m/z 1955.5 (Table 2). The rationale was that the +7 charge state of the ligand-bound forms of RNase A were above m/z 2000, which made +7 data incomplete and unusable (Table 3).\n\nThe +8 charge state used in this work is highlighted.\n\n1Where X=0 (no phosphate adduct), X=1 Pi (+98), X=2 Pi (+196), X=3 Pi (+294), X=4 Pi (+392), X=5 Pi (+490).\n\n1Where RNase A+dC5, L= +1383.9 (MWav) for one dC5, and RNase A+dC2AC2, L= +1408.0 (MWav) for one dC2AC2.\n\n2Where X=0 (no Pi adduct), X=1 Pi (+98), X=2 Pi (+196), X=3 Pi (+294), X=4 Pi (+392), X=5 Pi (+490).\n\nTo facilitate determination of total ion abundance, tables of predicted m/z values for free RNase A (Table 2) and the ligand-bound forms of RNase A (RNase A+dC5 and RNase A+dC2AC2) (Table 3) were constructed. A series of 98 Da adducts were included in Table 2 and Table 3 due to their presence in the mass spectra of this work, and that of earlier studies12,15. These adducts have been suggested to be either H2SO4 or H3PO418. Other RNase A studies have assigned these adducts as phosphate, and so each 98 Da adduct (X) in this work was designated as “Pi” (Table 2 and Table 3)12,15. Although mass spectra showed that free RNase A had up to 8 Pi adducts (Figure 1A and 1F), only the 0-5 Pi adduct forms of free RNase A and its ligand bound forms were used. This restraint was necessitated by the predicted m/z overlap of the ligand-bound forms of RNase A (with Pi adducts >5) with the m/z of free RNase at the +7 charge state. The “Qual Browser” feature of Xcalibur 1.4 SR1 software (Thermo) was used for analysis of each .raw file. For each sample, mass spectra comprising the two-minute data collection were averaged. The “spectrum list view” was used to obtain intensity data for all of the ions in the ranges comprising the +8 charge state (with 0-5 Pi adducts) for free RNase A (m/z 1710.7-1772.9), RNase A+dC5 (m/z 1883.7-1945.9), and RNase A+dC2AC2 (m/z 1886.7-1948.9). The intensity data for all ions in each m/z range were added to give the “total ion abundance” of the free (Ab(P)) and ligand-bound forms (Ab(PL)) of RNase A. The total ion abundance for the ligand-bound forms (RNase A+dC5 and RNase A+dC2AC2) were plotted as a function of [deoxyoligonucleotide] using GraphPad Prism 7.\n\nThe +8 charge state is shown. (A & F) no added deoxyoligonucleotide, (B) 5 μM dC5, (C) 10 μM dC5, (D) 20 μM dC5, (E) 40 μM dC5, (G) 5 μM dC2AC2, (H) 10 μM dC2AC2, (I) 20 μM dC2AC2, and (J) 40 μM dC2AC2. The number of phosphate adducts (Pi= 0-5) are indicated in four representative mass spectra (A, D, F, and I).\n\nThe total ion abundance ratio was determined at each [deoxyoligonucleotide] using the method described by Kitova et al.13, where for a 1:1 protein-ligand complex, the total ion abundance ratio (R) is calculated using the total abundance of all ligand-bound ions (Ab(PL)) and the total abundance of all free protein ions (Ab(P)) as shown in Equation 1:\n\nR= Ab(PL)/Ab(P) = [PL]eq/[P]eq [1]\n\nThe total ion abundance ratio (R) is used with the initial ligand concentration ([L]0) and initial protein concentration ([P]0) to calculate the association constant (Ka) using Equation 213:\n\nKa=R/([L]0 − ((R/(1+R))[P]0)) [2]\n\nThe Kd can then be calculated as the reciprocal of the Ka value.\n\n\nResults\n\nTable 1 indicates that samples contained an overall [RNase A] of 40.9 μM. Relatively low signal intensities observed for the +8 charge state of free and ligand-bound forms of RNase A necessitated this concentration, which was higher than the 5–20 μM RNase A used by others in nESI-Q-TOF-MS experiments12,15,16. Table 2 and Table 3 present predicted m/z values for free RNase A and the ligand-bound forms of RNase A (RNase A+dC5 and RNase A+dC2AC2) with multiple Pi adducts, which correlated well with observed m/z values (Figure 1). Upon increasing the concentration of dC5, the total ion abundance of free RNase A was found to decrease in intensity while the total ion abundance of RNase+dC5 was found to increase in intensity, which suggested 1:1 stoichiometry for the dC5:RNase A interaction (Figure 1A–E). Similar results were seen for the titration using dC2AC2 (Figure 1F–J). Table 4 presents total ion abundance data for free RNase A in samples that contained no added deoxyoligonucleotide. Total ion abundance data for free RNase A across six replicates gave a RSD of 16.4% (Table 4). Table 5 contains total ion abundance data for free RNase A and the ligand-bound forms of RNase A in samples that contained various concentrations of dC5 or dC2AC2. Total ion abundance data across three replicates at each [deoxyoligonucleotide] exhibited RSD values of approximately 20% or less (Table 5). A plot of the total ion abundance for free RNase A, RNase A+dC5, and RNase A+dC2AC2 as a function of [deoxyoligonucleotide] is shown in Figure 2. The total ion abundance for RNase A+dC5 and RNase A+dC2AC2 increased until 20 μM deoxyoligonucleotide, but decreased at 40 μM (Figure 2). Table 6 presents the calculated total ion abundance ratio (R) and dissociation constant (Kd) at each [deoxyoligonucleotide]. Samples containing <40 μM deoxyoligonucleotide unexpectedly produced negative Kd values (Table 6). By contrast, Table 6 shows that samples containing 40 μM deoxyoligonucleotide produced consistent positive values where the average Kd for dC5 was 2.2 ± 0.1 μM and the average Kd for dC2AC2 was 1.0 ± 0.1 μM.\n\nData is for the +8 charge state.\n\nData is for the +8 charge state.\n\n(A) [dC5] and (B) [dC2AC2]. The data is from Table 5, where points represent the average (n=3) ± standard deviation.\n\nData used for calculations was from Table 5.\n\n\nConclusions\n\nThis preliminary work demonstrates the potential and pitfalls of a LCQ ESI-IT-MS instrument to investigate protein-ligand interactions in an undergraduate teaching lab. Even though dC5 and dC2AC2 binding to RNase A are clearly illustrated in Figure 1, the presence of the Pi adducts complicated the mass spectra and broadened the signals for free RNase A and the ligand-bound forms of RNase A. In-source collision-induced dissociation was explored to reduce Pi adduct formation, but it appeared to disrupt the RNase A+dC5 and RNase A+dC2AC2 complexes, and so this approach was abandoned (data not shown). It is unclear why the decrease in the total ion abundance for the ligand-bound forms of RNase A was observed at higher deoxyoligonucleotide concentrations (Figure 2). Previously, the ion intensity ratio of free RNase A to the RNase A+cytidine 2′-monophosphate (2′-CMP) complex was observed to vary with charge state as follows: +8 (0.65), +7 (0.73), +6 (1.1)12. This led Zhang et al. to suggest that either the binding of ligand, or the presence of ligand in the analyzed RNase A samples, created a change of the charge state distribution for the protein-ligand complex12. In the present work, the binding of deoxyoligonucleotide, or the presence of deoxyoligonucleotide in samples, could have shifted some of the total ion abundance of free and/or ligand-bound RNase A from the +8 charge state to lower charge states, which were beyond the mass range of our ion-trap mass analyzer. This highlights an inherent limitation of this work, which was the inability to gather data for all free and ligand-bound RNase A charge states. Kitova et al. stated the importance of including all ligand-bound and free protein ions in the calculation of R, and emphasized that the “sometimes-used practice” of employing a particular charge state to determine Ka should be avoided13. Thus, the lack of data for the +7 and +6 charge states of RNase A hindered accurate collection of total ion abundance data, which may have affected calculations of R and led to the negative Kd values at low ligand concentrations (Table 6). The positive Kd values in Table 6 are of similar magnitude to those determined by Zhang et al. for 2′-CMP and CTP, via a nESI-Q-TOF-MS titration experiment, which were 1.7 ± 0.3 μM and 0.8 ± 0.2 μM, respectively12. They are also in the neighborhood of in solution Kd measurements (3-24 μM) observed for the binding of short fluorescein-labeled deoxyoligonucleotides to RNase A19. In conclusion, while RNase A is an excellent model for many experiments, instructors wishing to use a LCQ ESI-IT-MS instrument to investigate protein-ligand interactions are encouraged to consider other protein-ligand systems that would enable all charges states (of the free and ligand-bound protein) to be observed.\n\n\nData availability\n\nDataset 1. LCQ .raw data files for all samples. 10.5256/f1000research.14268.d19837320\n\nData files 1–6 are for samples that contained free RNase A (6 replicates), Data files 7–18 are for samples that contained RNase A and dC5 (3 replicates per [dC5]), Data files 19–30 are for samples that contained RNase A and dC2AC2 (3 replicates per [dC2AC2]).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the College of Natural Sciences and the Department of Chemistry and Biochemistry at California State University- Chico.\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\nI thank Professor Daniel Edwards at California State University-Chico for helpful discussions and review of the manuscript.\n\n\nReferences\n\nMarshall GR, Feng JA, Kuster DJ: Back to the future: ribonuclease A. Biopolymers. 2008; 90(3): 259–277. PubMed Abstract \| Publisher Full Text\n\nPress Release: The 1972 Nobel Prize in Chemistry. 1972, (accessed March 1, 2018). Reference Source\n\nWahida F, Shehzada A, Khan T, et al.: MicroRNAs: synthesis, mechanism, function, and recent clinical trials. Biochim Biophys Acta. 2010; 1803(11): 1231–1243. PubMed Abstract \| Publisher Full Text\n\nCuchillo CM, Nogués MV, Raines RT: Bovine pancreatic ribonuclease: fifty years of the first enzymatic reaction mechanism. Biochemistry. 2011; 50(37): 7835–7841. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCounterman AE, Thompson MS, Clemmer DE: Identifying a Protein by MALDI-TOF Mass Spectrometry: An Experiment for the Undergraduate Laboratory. J Chem Educ. 2003; 80(2): 177–180. Publisher Full Text\n\nReimann CT, Mie A, Nilsson C: Introduction to biological mass spectrometry: Determining identity and species of origin of two proteins. J Chem Educ. 2005; 82(8): 1215–1218. Publisher Full Text\n\nAlbright JC, Dassenko DJ, Mohamed EA, et al.: Identifying gel-separated proteins using in-gel digestion, mass spectrometry, and database searching: Consider the chemistry. Biochem Mol Biol Educ. 2009; 37(1): 49–55. PubMed Abstract \| Publisher Full Text\n\nShort M, Short A, Vankempen R, et al.: Using HPLC-mass spectrometry to teach proteomics concepts with problem-based techniques. Biochem Mol Biol Educ. 2010; 38(4): 242–246. PubMed Abstract \| Publisher Full Text\n\nWilson KA, Tan-Wilson A: Seed storage proteins as a system for teaching protein identification by mass spectrometry in biochemistry laboratory. Biochem Mol Biol Educ. 2013; 41(2): 79–86. PubMed Abstract \| Publisher Full Text\n\nAlty LT, LaRiviere FJ: Peptide mass fingerprinting of egg white proteins. J Chem Educ. 2016; 93(4): 772–777. Publisher Full Text\n\nBoeri Erba E, Petosa C: The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes. Protein Sci. 2015; 24(8): 1176–1192. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang S, Van Pelt CK, Wilson DB: Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry. Anal Chem. 2003; 75(13): 3010–3018. PubMed Abstract \| Publisher Full Text\n\nKitova EN, El-Hawiet A, Schnier PD, et al.: Reliable determinations of protein-ligand interactions by direct ESI-MS measurements. Are we there yet? J Am Soc Mass Spectrom. 2012; 23(3): 431–441. PubMed Abstract \| Publisher Full Text\n\nIshii K, Noda M, Uchiyama S: Mass spectrometric analysis of protein-ligand interactions. Biophys Physicobiol. 2016; 13(2016): 87–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSundqvist G, Benkestock K, Roeraade J: Investigation of multiple binding sites on ribonuclease A using nano-electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom. 2005; 19(8): 1011–1016. PubMed Abstract \| Publisher Full Text\n\nYin S, Xie Y, Loo JA: Mass spectrometry of protein-ligand complexes: enhanced gas-phase stability of ribonuclease-nucleotide complexes. J Am Soc Mass Spectrom. 2008; 19(8): 1199–1208. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBirdsall DL, McPherson A: Crystal structure disposition of thymidylic acid tetramer in complex with ribonuclease A. J Biol Chem. 1992; 267(31): 22230–22236. PubMed Abstract\n\nChowdhury SK, Katta V, Beavis RC, et al.: Origin and removal of adducts (molecular mass = 98 u) attached to peptide and protein ions in electrospray ionization mass spectra. J Am Soc Mass Spectrom. 1990; 1(5): 382–388. PubMed Abstract \| Publisher Full Text\n\nFisher BM, Grilley JE, Raines RT: A new remote subsite in ribonuclease A. J Biol Chem. 1998; 273(51): 34134–34138. PubMed Abstract \| Publisher Full Text\n\nClark DD: Dataset 1 in: Preliminary investigation of deoxyoligonucleotide binding to ribonuclease A using mass spectrometry: An attempt to develop a lab experience for undergraduates. F1000Research. 2018. Data Source" }	[ { "id": "32248", "date": "09 Apr 2018", "name": "Samuel J. Allen", "expertise": [ "Reviewer Expertise Native Mass Spectrometry" ], "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\nGeneral Comments: This work describes an experimental approach to introduce native mass spectrometry to undergraduate students. The author provides sufficient context for the choice of Ribonuclease A as the target protein, and describes the use of mass spectrometry under “native” conditions. This work also introduces undergraduates to practical use of mass spectrometry by providing “expected” m/z tables and shows the effect of adducted species on mass spectrometry signal. Plainly stated, the proposed experiment did not meet the initial hypothesis of the author. One major limitation to this study was the use of an ion trap mass spectrometer, which the author states as being the only available instrument at the academic institution. Additionally, the data from this study results in negative dissociation constant values for the lowest ligand concentrations, but reasonable dissociation constants at the highest ligand concentration. The author attempts to describe these results relative to other similar studies. As a result, this work describes the “potential and pitfalls” of attempting this experiment, which is useful for undergraduate students that are early in their scientific career.\n\nSuggestion (no change requested): For future investigations and undergraduate studies, it would be beneficial to use centrifugal desalting columns in an attempt to remove the phosphate adducts. This would result in improved ion response, less convoluted spectra, and would introduce undergraduate students to common sample preparation used in native-like protein MS experiments.\n\nSuggestion (no change requested): For future investigations and undergraduate studies, a native MS technique that has been used to address the upper m/z limitation of ion trap is to “supercharge” proteins1. This can be done by adding as low as 1% v/v sulfolane or m-nitrobenzyl alcohol to the sample.\n\nAdditional comments to conclusion (minor revisions requested): There are two issues that the authors addresses regarding the data from this study. (1) The observation of negative dissociation constants and (2) decreasing PL abundance at the highest L concentration. To point (1), although these experiments are being performed under native conditions (i.e. non denaturing solvents), there are still other factors during electrospray ionization that need to be considered during native experiments. For example, Benkestock, et al2 show data that suggests that the “capillary-to-cone” distance and the electrospray probe internal diameter can affect the PL to L ratios. To point (2), the decrease in PL abundance at the highest L concentration may be due to non-specific binding as described in the already cited Kitova et al. (2012)3(Section 2.4 and Figure 3). The author should add a couple sentences to the conclusion addressing how “non-ideal” ionization conditions and non-specific binding could have affected the measurements.\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": [] }, { "id": "32249", "date": "12 Apr 2018", "name": "Ryan N. Jackson", "expertise": [ "Reviewer Expertise Biochemistry", "Structural 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\nIn this manuscript, author Dan Clark describes an experimental protocol aimed at introducing undergraduate biochemistry students to mass spectrometry methods. Native electrospray ion-trap mass spectrometry (ESI-IT-MS) was used to produce mass spectra of unbound ribonuclease A (RNase A) and ligand – bound RNase A at differing concentrations of added oligonucleotides. Dissociation constants were determined using observed abundance ratios of bound and unbound RNAse A. However, dissociation constants with small concentrations of oligonucleotides were inaccurate, while the largest concentration produced a dissociation constant similar to that previously published. The author explains that these discrepancies likely result from limitations in the mass spectrometry equipment available, and concludes that a different protein – ligand combination may be better suited for the desired experiment.\n\nOverall this was a well-conducted investigation and meets an acceptable standard for publication. Contradictory to the conclusion of the author that another protein – ligand combination may be better suited to teach undergrads about mass spec, I found strong educational merit in the failures of this protocol to determine all dissociation constants. As an instructor of undergraduates I have found that students can often learn more when things do not work exactly as expected. The experiment presented here offers an opportunity for students to understand equipment limitations and may help students obtain a stronger understanding of how the diversity of charged states impacts the ability to collect an accurate mass spectra.\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/7-340
https://f1000research.com/articles/7-497/v1	26 Apr 18	{ "type": "Research Article", "title": "TDG regulates cell cycle progression in human neural progenitors", "authors": [ "Igal Germanguz", "Jenny C. Park", "Jessica Cinkornpumin", "Aryeh Solomon", "Minori Ohashi", "William E. Lowry", "Igal Germanguz", "Jenny C. Park", "Jessica Cinkornpumin", "Aryeh Solomon", "Minori Ohashi" ], "abstract": "Background: As cells divide, they must both replicate their DNA and generate a new set of histone proteins. The newly synthesized daughter strands and histones are unmodified, and must therefore be covalently modified to allow for transmission of important epigenetic marks to daughter cells. Human pluripotent stem cells (hPSCs) display a unique cell cycle profile, and control of the cell cycle is known to be critical for their proper differentiation and survival. A major unresolved question is how hPSCs regulate their DNA methylation status through the cell cycle, namely how passive and active demethylation work to maintain a stable genome. Thymine-DNA glycosylase (TDG), an embryonic essential gene, has been recently implicated as a major enzyme involved in demethylation. Methods: We use human pluripotent stem cells and their derivatives to investigate the role of TDG in differentiation and proliferation. To perform loss of function of TDG, RNA Interference was used. To study the cell cyle, we engineered human pluripotent stem cells to express the FUCCI tool which marks cells at various stages of the cell cycle with distinct patterns of fluorescent proteins. We also used cell cycle profiling by FACS, and DNA methylation analysis to probe a connection between DNA demethylation and cell cycle. Results: Here we present data showing that TDG regulates cell cycle dynamics in human neural progenitors (NPCs) derived from hPSCs, leading to changes in cell cycle related gene expression and neural differentiation capacity. These data show that loss of TDG function can block differentiation by driving proliferation of neural progenitors. We also identify specific cell cycle related genes whose expression changes upon loss of TDG expression. Conclusions: These observations suggest that TDG and active demethylation play an important role in hPSC cell cycle regulation and differentiation.", "keywords": [ "TDG", "DNA methylation", "human neural progenitors" ], "content": "Introduction\n\nCoordinated changes to the epigenome are known to be essential for lineage specification and maintenance of cellular identity. DNA methylation and histone modifications critically contribute to epigenetic maintenance of chromatin structures and gene expression programs. DNA methylation can silence genomic regions, directly or indirectly, and play an important role during mammalian development. Loss of methylation in specific locations is associated with differentiation towards specific germ layers as binding of several transcription factors is strongly associated with specific loss of DNA methylation in one germ layer, and in many cases a reciprocal gain in the other layers. However, the mechanism for the lineage related site specific demethylation is not currently known. A major open question is whether this is the result of an active or passive demethylation after repeated cell division1,2. Promoters with low CpG content are more likely to be methylated in human embryonic stem cells (ESCs). Conversely, these same promoters are demethylated and actively expressed during differentiation in a cell-type-specific manner3–5. Demethylation can occur by a passive mechanism in which the normal function of DNMT1/UHRF1 is insufficient or disrupted2,6. Alternatively, evidence for the existence of an active mechanism in which the cytosine modifications are enzymatically removed is accumulating1,7–9. Which of these mechanisms is responsible for demethylation changes in early human development is not currently known.\n\nWe and others have shown that standard differentiation protocols of hPSC leads to derivation of an embryonic-like cell rather than a mature, postnatal-like cell10. We previously identified a group of embryonic related genes which are differentially expressed in the PSC progeny of all three lineages and in tissues of the early gestation period rather than in their respective cell types of later developmental stages. Among these genes, we identified Thymine DNA Glycosylase (TDG), a gene that was recently implicated in active DNA demethylation10. Unlike other glycosylases, TDG is essential for embryonic viability as TDG null embryos die around E11.5-12.5 of internal hemorrhage8. The lethal phenotype was also associated with aberrant promoter methylation and imbalanced histone modifications. In addition there is evidence that levels of this enzyme are linked to progression through specific cell cycle stages. Here we provide evidence that that TDG regulates cell cycle related gene expression in human neural progenitors (NPCs) derived from hPSCs and controls their capacity for differentiation towards neurons and glia. These observations suggest that TDG and active demethylation play an important role in hPSC cell cycle regulation and differentiation.\n\n\nResults\n\nWe originally identified TDG in a screen for genes that were consistently differently expressed between human pluripotent derivatives and their in vivo counterparts10. This screen identified a number of genes that were persistently expressed in pluripotent derivatives, and therefore suggestive of an early embryonic state, or genes that failed to be induced in pluripotent derivatives but were highly expressed in tissue derived cells. TDG was expressed significantly higher in neural progenitors generated from pluripotent stem cells as opposed to the same cell type derived from fetal brain (Figure 1A, shown in Log2 scale).\n\nA, Average expression of the TDG microarray probe in hPCS, hPSC derived NPCs and neurons and tissue derived NPCs, adapted from 10. Data presented are the average of at least three independent samples and normalized by log2 according to 13. B, RNA-seq analyses for the expression of TDG, TET1, 2 and 3 adopted from the Allen Institute's Brainspan developmental transcriptome database displayed as log-scale reads per kilobase measured (log2 RPKM) across the developing human brain. C, Immunofluorescent (IF) staining for KI67 and TDG expression in NPCs derived from human pluripotent stem cells (PSC-NPCs) or human brain tissue (Tissue-NPCs). D, Magnified images (from panel C) of representative nuclei for different cell cycle stages. Images were taken at 20X magnification.\n\nThe Allen Brain Atlas created by the Allen Institute provides gene expression data from various brain regions across both development and through adulthood. As shown in Figure 1B, TDG is expressed most highly in the brain in utero, and then falls after birth and stays low throughout adulthood. The same was true for the Ten–Eleven Translocation (TET) family of dioxigenases7, suggesting that DNA demethylation is primarily performed in utero. It is also possible that DNA demethylation by TDG and TETs function is linked to proliferation, which is known to decrease at birth relative to that found in utero. Because of this and previous data suggesting TDG could potentially regulate the cell cycle, we stained neural progenitors made from human pluripotent stem cells or derived from tissue for TDG and Ki67. The cell cycle phase for this assay was determined based on a previously reported KI67 staining pattern within the nucleus11. TDG was also previously reported to be tightly regulated during the progression of the cell cycle as its level is rapidly downregulated by ubiquitination in the S phase of the cell cycle in cellular models such as HeLa and fibroblasts and re-expressed in G212. Here, we found a similar result, namely that TDG protein levels appear to correlate with G0/G1stages of the cell cycle (Figure 1C and D).\n\nTo investigate the role of TDG in early human development, we used siRNA mediated knock-down (KD) of TDG in neural progenitor cells (NPCs) derived from hPSC (Figure 2A and B). To determine whether TDG KD led to expected changes in 5-carboxylcytosine (5caC) and 5-formylcytosine (5fC) DNA residues, immunostaining for these markers was performed. As expected, silencing TDG led to an increased intensity of 5caC and 5fC DNA, with no change in 5-Hydroxymethylcytosine (5hmC) DNA (Figure 2C).\n\nA, TDG protein expression level at day 4 post siRNA transfection, measured by Western blot (top). B, Immunostaining for TDG in NPCs following siRNA transfection, and quantification (right). C, Top: Representative immunofluorescence of DNA methylation modifications. Bars represent 50µm. Bottom: ImageJ quantification of TDG-KD normalized to CONT-KD. Quantification was performed for over 100 nuclei across at least 5 images. Error bars represent standard error of the mean normalized to CONT-KD. D, Expression levels of NPC markers measured by qRT-PCR, normalized against the relative levels of GAPDH and compared to CONT-KD, error bars represent standard error of the mean of 3 knockdown experiments. E, 4 Days post siRNA transfection, NPCs were induced to terminally differentiate by growth factor withdrawal (GFWD). Left: representative IF of 3 Weeks neural differentiation. Efficiency measured by the ratio of percentage of MAP2 (neuron)/GFAP (glia). Bars represent 50µm. Right: quantification of n = 3 from at least three separate knockdown/differentiation experiments. p-values were calculated with Student's t test: * = p<0.05, ns=not significant.\n\nRT-PCR for genes typical of the NPC state showed essentially no change in TDG KD cells (Figure 2D). Though downregulating TDG levels showed no influence on NPC identity, we further tested whether lower TDG levels affects differentiation. Four days post TDG KD treatment, NPCs were induced to further differentiate using the growth factor withdrawal method (removal of self-renewal supporting growth factors EGF, bFGF). Three weeks after induction of differentiation, we analyzed the percentage of MAP2/GFAP positive cells, which represent the differentiation expectancy towards the neuronal/glial lineage respectfully. We found that though the neural/glial ratio remained similar, the total differentiated cell percentage was lower than in control (Figure 2E), indicating a failure to properly differentiate upon silencing of TDG. Typically, such a differentiation block would be due to aberrant differentiation or due to prolonged proliferative stimulus.\n\nWe also looked for gene expression changes following TDG-KD in NPCs by RNA-SEQ. 355 genes were differentially expressed by 1.5 fold across 3 independent experiments (Figure 3A). Using the DAVID annotation tool14, we classified those genes into functional groups (Figure 3B). Of the most significantly enriched functional annotations identified, we found 34 cell cycle related genes. Among those, genes which are major players in mitosis, CDK1, CDK10, SKP2 were upregulated. In contrast, other genes like CDC25B and CDKN1C (p57), which are inhibitors of cell cycle progression, were downregulated (Figure 3C).\n\nA, Differential gene expression of n = 3 siRNA knockdown experiments. Scatter plot of the group average FPKM (log2) for all genes mapped above the background cutoff, differentially expressed genes (over 1.5 fold change; p<0.05) are highlighted in red and green. B, Functional annotation of differentially expressed genes shows significant change in genes related to cell cycle, regulation of apoptosis and structural genes. C, Cell Cycle related differentially expressed list of genes and the relative fold change.\n\nTo validate that silencing of TDG by siRNA led to changes in DNA demethylation, we performed Methylase-assisted bisulfite conversion PCR (MAB-PCR) to probe for the presence of the 5mC and 5hmC in a gene whose expression changed upon siRNA-mediated knockdown of TDG. MAB-PCR takes advantage of an enzyme and bisulfite-conversion sequencing to identify the relative abundance of 5caC and 5fC nucleotides (Figure 4A). This allows for a measure of TDG activity, as TDG is known to use its glycosylase activity to finish the demethylation process to convert 5caC and 5fC to the fully demethylated state. To determine whether TDG activity can regulate the methylation and gene expression, we looked specifically at a gene whose expression was affected in TDG-KD cells. EGR1, the early growth response gene is known to be dynamically regulated by a variety of mechanisms, including DNA Methylation at an upstream CpG island (Figure 4B)15. We analyzed a segment of a CpG island upstream to the EGR1 transcription start site (TSS site). This locus was chosen as it was reported to have the highest distribution of 5fC, 5caC around the TSS.\n\nA, Schematic illustration of the sequencing of methylase treated compared to non-treated bisulfite converted transcripts. B, EGR1 expression levels are downregulated following TDG knockdown as measured by RNA-seq (described in Figure 2). C, Sanger sequencing of the CpG island upstream to the EGR1 TSS following either bisulfite conversion (BS only; top) or MAB treatment (bottom two) shows higher abundance of 5cAC, 5fC in TDG deficient cells; numbers indicate CpG dinucleotide position. D, Summary visualization (left) and quantification (right) of the abundance of non-converted residues described in Figure 4C. Error bars represent standard error of the mean methylation level of at least 5 sequenced samples, p values were calculated with Student's t test: * = p<0.001.\n\nThis analysis showed that silencing of TDG by siRNA led to a dramatic accumulation of 5caC and 5fC in a CpG island directly upstream of the start site of EGR1 transcription (Figure 4C and D). This experiment provided evidence that TDG not only regulates DNA demethylation, but also that this can influence gene expression. The proportion of genes differentially regulated by TDG-mediated DNA demethylation remains unclear until a genome-wide analysis can be performed.\n\nWe further tested whether TDG-KD in NPCs affects entrance into the cell cycle by Ki67 staining, and found that downregulating TDG resulted in a higher percentage of proliferating cells when this enzyme was knocked down (Figure 5A). Co-staining of Ki67 with TDG showed that TDG is downregulated with cell cycle progression, as higher TDG expression is observed in G0/early G1 cells, and downregulated with cell cycle progression (Figure 2D). We also measured cell cycle dynamics by Flow Cytometry (FACS) upon TDG silencing. This high-throughput method allowed for an accurate determination of the effect of siRNA on TDG, and showed that the proportion of cells in S phase was significantly decreased, while the proportion in G2/M was increased (Figure 5C). Taken together, it seems clear that TDG plays a role in human pluripotent stem cell cycle regulation.\n\nA, TDG downregulation results in higher fraction of cells entering mitotic cell cycle, based on KI67 positive cells staining compared to CONT-KD. Left: representative IF (images were taken at 10X magnification). Right: quantification of over 400 cells from five separate fields. Error bars represent standard error of the mean. p-values were calculated with Student's t test: =p<0.01. B, TDG downregulation results with a change in cell cycle progression as G2/M increased. Left: representative flow analysis (out of four independent TDG-KD experiments analyzed by flow cytometry) of TDG-KD NPC cell cycle compared to CONT-KD NPC, based on DNA content staining with PI. Right: Quantification of cell cycle phases from 4 separate TDG knockdown experiments\n\nDespite all the analyses above, it was not clear whether silencing of TDG affects the cell cycle through its ability to regulate the terminal step of DNA demethylation. It is formally possible that the DNA glycosylation activity of TDG is used for other substrates besides methylated cytosine, for instance in DNA repair. It is also possible that another domain of TDG regulates cell cycle progression by another unknown mechanism. To attempt to link DNA demethylation by TDG to regulation of the cell cycle, we needed a system that could allow for simultaneous labeling of DNA demethylation intermediates and cell cycle markers. We generated hESCs which expressed the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgene reporter system by lentiviral transduction16. In this system, cells which are in the G1 stage express Ctd1, which is conjugated to mCherry, while cells in S/G2 expressed Geminin which is conjugated to visible green protein mVENUS. Cells entering the DNA replication stage at the end of G1 express both markers and emit yellow light (Figure 6A). We first verified that the level of TDG was tightly regulated during cell cycle progression in hPSC as in other reported cell systems since hPSC display a unique cell cycle pattern. We found high levels of TDG in early G1 which are downregulated with cell cycle progression (Figure 6B). Interestingly, we found that 5caC and 5fC were both induced in early S phase cells, while 5hmC was reduced (Figure 6B) as was reported before. This indicates that the global state of DNA demethylation is tightly correlated to progression of the cell cycle. Furthermore, all these data on the effect of TDG on cell cycle serve to explain why silencing of TDG led to defective neuronal and glial specification in NPCs (Figure 2D).\n\nA, Illustration of the FUCCI cell cycle reporter system expression. B, Co-staining for particular cell cycle phase with antibody against either TDG or methylation intermediate modifications. Left: representative nucleotide for each cell cycle stage (Pics were taken using a 20X magnification and a cropped single nucleotide is presented) Right: intensity of staining was quantified using ImageJ for over 500 cells, values normalized to G1 (Y Axis) are presented. p values were calculated with Student's t test: =p< 0.01, *=p<0.001, ns=not significant.\n\n\nDiscussion\n\nThe data presented here confirm and extends previous findings that TDG and DNA demethylation can play a role in proper progression through the cell cycle. These results could be particularly relevant for the nervous system, where we provide evidence that TDG and TET mediated demethylation appears to diminish across development. This correlates with both proliferative rate and TDG expression, and could have important consequences to the rate of developmental progression. The big question remaining from this work is how DNA demethylation plays a role in progression of the cell cycle. When DNA is replicated it is thought that the new daughter strand is methylated according to the hemi-methylation pattern on the sister strand by maintenance DNA Methyltransferases (DNMT1). Less clear is what happens to portions of the genome that are hemi-methylated hydroxymethylated nucleotides. The change in proportions of 5caC and 5fC across the cell cycle could indicate that these modified nucleotides are simply erased through the action of TET and TDG enzymes, and then re-written. In this scenario, it is interesting that blocking TDG appeared to promote the cell cycle rate, and could suggest that demethylation is a rate limiting step in cell cycle progression to ensure proper methylation of DNA in both daughter cells.\n\nBecause of the difference in expression levels of TDG between pluripotent and tissue derived NPCs (Figure 1A), we expected that silencing TDG would have a positive effect on the progression of developmental maturity of the NPCs. The LIN28/let-7 circuit was previously shown to be differentially regulated between NPCs born from pluripotent stem cells versus those derived from tissues, and resolution of this discrepancy was sufficient to advance the developmental maturity of NPCs in that context17. When the expression of TDG was brought down to a level similar to that seen in tissue derived NPCs, instead of advancing developmental maturation, the cells appeared to be unable to efficiently differentiate (Figure 2). This was presumably due to the increased rate of proliferation of the NPCs, a cell type where forced exit of the cell cycle is known to induce differentiation. Therefore, experimentally regulating TDG levels does not facilitate differentiation from pluripotent stem cells, as was the case with experimental downregulation LIN28. Perhaps the more interesting result from this work is that pluripotent derivatives probably need to silence TDG expression or activity at a more developmentally appropriate time point to proceed through proper development.\n\n\nMethods\n\nH9 hESCs and XFIPS2 were used in this study in accordance with the UCLA Embryonic Stem Cell Research Oversight committee (ESCRO, 2006-019-11A) and the Institutional Biosafety Committee (IBC). This work was specifically described in our ESCRO application (2006-019-11A), and approved on an annual basis by the committee. This work is considered not human subjects research by the UCLA IRB.\n\nCells were cultured in feeder free conditions on Matrigel (Corning) using mTeSR1 (Stem Cell Technologies) and passaged mechanically or with collagenase every 4–5 days. NPCs differentiation was performed as described previously (10). Briefly, for rosette induction, 80% confluent hPCS were cultured in DMEM/F12 with N2 and B27 supplements (Invitrogen), 20 ng/ml basic fibroblast growth factor (bFGF; R&D Systems), 1 µM retinoic acid (Sigma), 1 µM Sonic Hedgehog Agonist (Purmorphamine; Sigma), 10µM SB431542 (TGFß inhibitor; Cayman) and 0.1µM LDN193189 (BMP receptor type 1 inhibitor; Cayman). Small molecules were resuspended according to each manufacturer instructions. After about a week of culture neural-like rosettes were mechanically picked and expanded in NPC maintenance medium: DMEM/F12 supplemented with N2/B27, bFGF and 500 ng/ml epidermal growth factor (EGF; GIBCO). For further differentiation, the growth factors bFGF and EGF were withdrawn from the media (GFW) and cultured for 3 weeks. TDG and control knockdown in NPCs was performed using a unique 27-mer siRNA duplexes (Trilencer, Origene) at a final concentration of 20 nM using Lipofectamine RNAiMAX transfection reagent (Invitrogen).\n\nImmunofluorescent staining was performed using standard protocol10,17. Briefly, cover slips were fixed with 4% PFA in PBS for 20 min, washed and then permeabilized and blocked in 10% donkey serum, 0.01% Triton in PBS for 1 hour. Primary antibodies in 5% donkey serum were incubated for 1–2 hours at room temperature following 3X wash in PBST and incubation with conjugated secondary antibody for 1 hour in room temperature. After 3X wash with PBST cover slips were incubated with 300nM DAPI final concertation in PBST for 3 min in room temperature (dark), followed with 3X wash with PBST. Antibodies used include the following: polyclonal rabbit anti-TDG 1:100(Atlas; HPA052263); polyclonal chicken anti-GFAP 1:1000(Abcam; ab4674); mouse monoclonal anti-MAP2 1:500 (Abcam, ab11267), polyclonal rat anti-KI67 1:100 (eBioscience; 14-5698). For methylation modifications, permeabilized cells were denatured with 2N HCl for 15 min and then neutralized with 100 mM Tris-HCl (pH 8.5) for 10min before blocking. The following Active-Motif Antibodies were used: rabbit anti 5hmC 1:100(39770); rabbit anti-5fC 1:2500 (61223); rabbit anti-5caC 1:1000 (61225). Image analysis and quantification was performed using ImageJ version 1.50i with the same threshold for each channel for all samples. Western Blot analysis was performed using standard procedures as described (Lowry et al., 2005) antibodies used were rabbit anti-TDG as described above and mouse anti-Actin 1:1000 (Santa Cruz Biotechnology, sc-47778).\n\nRT-PCR Analysis. Total RNA was extracted using an RNeasy Mini Kit (QIAGEN). cDNA synthesis was performed using the Superscript III first-strand cDNA synthesis kit (Invitrogen). Real-time PCR was performed in triplicate using the SYBR green real-time PCR MIX (Roche) in the Roche lightcycler 480 machine. Run was performed for 50 cycles and analysis was performed in Microsoft Excel 2013 using the 2-ΔΔCT method.\n\nTotal RNA was extracted using an RNeasy Mini Kit (QIAGEN). Libraries were constructed according to manufacturer instructions (TruSeq Stranded Total RNA with Ribo-Zero; Illumina). Following second strand PCR amplification, ~200bp sized libraries were excised from agarose gel and pooled together in 10mM concentration each. Samples were sequenced using Illumina HiSeq2000 on single-end 50-bp reads and aligned to human reference genome (Hg19) using Tophat (version 2.0.6)18. Processing using Cufflinks and Cuffdiff was performed to obtain differential fragments per kilobase of transcript per million mapped reads (FPKM)18. Three biological replicates (i.e. 3 separate knockdown experiments in different PSC clones) were grouped together. Further analysis was performed using the cummeRbund suite (v2.0.0). Functional annotation was performed using DAVID (V6.7)14.\n\nFollowing trypsin dissociation, knocked-down NPCs were fixed overnight in 70% ethanol at -20°C. Fixed cells were then stained for half an hour at room temperature in the dark, with Propidium Iodide (PI) for a final concertation of 50 µg/ml supplemented with RNAse (final 1 µg/ml). DNA content was analyzed on BD-Biosciences LSR-II flow cytometer and cell cycle phases were determined using the FlowJo cell cycle module (version 7.6.5).\n\nGenomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen; 69506). One µg genomic DNA was treated by M.SssI (New England Biolabs; M0226s) in a 50 µl reaction for three rounds. For each round DNA was incubated with 4 Units of M.SssI CpG methyltransferase (NEB), supplemented with 160 mM final S-Adenosyl methionine for 3 hours at 37°C. At the end of each round DNA was cleaned using phenol/chloroform extraction. Bisulfite conversion was performed using the EpiTect Bisulfite Kit (QIAGEN; 59104) and then selected loci was PCR amplified with KAPA HiFi Hotstart Uracil+ DNA polymerase (KAPABiosystems). The resulting PCR product was cloned into the TOPO-Blunt (Invitrogen) vector, and sent to Laragen for Sanger sequencing (GeneWiz). Analysis and visualization of sequence reads was done using the online BISMA tool.\n\nThe FUCCI reporter lentiviral plasmids, pCSII-EF-mCherry-hCdt1(30/120) and pCSII-EF-mVenus-hGeminin(1/110) were a generous gift of Dr. Atsushi Miyawaki (RIKEN Brain Science Institute, Saitama, Japan). Lentiviral virions were generated in 293T cells using standard protocols as previously described13 followed by concentration with Amicon Ultra-15 centrifugal units (100K; Millipore). hPSC were single celled with TryplE (Thermo) and re-plated 24h prior to infection supplemented with 10 µM Rho-associated kinase (ROCK) inhibitor Y27632 (Stemgent). Cells were first infected with one reporter lentivirus particles for overnight infection. Cells were washed with fresh medium and grown for 2– 3 passages for recovery and expansion. Next, we FACS sorted using FacsARIA (Becton Dickinson) the cells for the corresponding reporter to ensure that all cells are infected. Briefly: cells from a whole 6 well plate were treated with ROCK inhibitor for one hour, single celled using trypLE and re-suspended in PBS. After sorting cells were a re-plated for recovery for 5–7 days and then\n\nWe infected the cells with the second reporter using that same procedure followed by a second FACS sorting for the reporter.\n\n\nStatistical analysis\n\nStudent’s t-test was performed using GraphPad 6.01. Results were judged to be significant if the p-value was < 0.05. All other statistical analysis described in this study were performed using Microsoft Excel 2013\n\n\nData availability\n\nDataset 1: TDG regulates cell cycle progression in human neural progenitors. Complete dataset of all underlying data divided into folders based on relevant figures. 10.5256/f1000research.13801.d20137919", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by NIH (P01GM9913), Allen Distinguished Investigator award from the Allen Frontiers Group to WEL, A CIRM Basic Biology Award (RT-2), and pilot support from the BSCRC at UCLA (Rose Hills Scholar Award).\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 the support of various core facilities and their staff at the core facilities sponsored by the Eli and Edythe Broad Center for Regenerative Medicine (EEBCRC) including: Flow Cytometry, Genomics, and the Stem Cell Cores.\n\n\nReferences\n\nKohli RM, Zhang Y: TET enzymes, TDG and the dynamics of DNA demethylation. Nature. 2013; 502(7472): 472–479. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKristensen DG, Nielsen JE, Jørgensen A, et al.: Evidence that active demethylation mechanisms maintain the genome of carcinoma in situ cells hypomethylated in the adult testis. Br J Cancer. 2014; 110(3): 668–678. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKinney SR, Pradhan S: Ten eleven translocation enzymes and 5-hydroxymethylation in mammalian development and cancer. Adv Exp Med Biol. 2013; 754: 57–79. PubMed Abstract \| Publisher Full Text\n\nCortázar D, Kunz C, Selfridge J, et al.: Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability. Nature. 2011; 470(7334): 419–423. PubMed Abstract \| Publisher Full Text\n\nRai K, Huggins IJ, James SR, et al.: DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd45. Cell. 2008; 135(7): 1201–1212. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatterson M, Chan DN, Ha I, et al.: Defining the nature of human pluripotent stem cell progeny. Cell Res. 2012; 22(1): 178–193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBraun N, Papadopoulos T, Müller-Hermelink HK: Cell cycle dependent distribution of the proliferation-associated Ki-67 antigen in human embryonic lung cells. Virchows Arch B Cell Pathol Incl Mol Pathol. 1988; 56(1): 25–33. PubMed Abstract \| Publisher Full Text\n\nWheldon LM, Abakir A, Ferjentsik Z, et al.: Transient accumulation of 5-carboxylcytosine indicates involvement of active demethylation in lineage specification of neural stem cells. Cell Rep. 2014; 7(5): 1353–1361. PubMed Abstract \| Publisher Full Text\n\nGermanguz I, Listgarten J, Cinkornpumin J, et al.: Identifying gene expression modules that define human cell fates. Stem Cell Res. 2016; 16(3): 712–724. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang da W, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009; 4(1): 44–57. PubMed Abstract \| Publisher Full Text\n\nPenner MR, Parrish RR, Hoang LT, et al.: Age-related changes in Egr1 transcription and DNA methylation within the hippocampus. Hippocampus. 2016; 26(8): 1008–1020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingh AM, Trost R, Boward B, et al.: Utilizing FUCCI reporters to understand pluripotent stem cell biology. Methods. 2016; 101: 4–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatterson M, Gaeta X, Loo K, et al.: let-7 miRNAs can act through notch to regulate human gliogenesis. Stem Cell Reports. 2014; 3(5): 758–73. 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\n\nGermanguz I, Park J, Cinkornpumin J, et al.: Dataset 1 in: TDG regulates cell cycle progression in human neural progenitors. F1000Research. 2018. Data Source" }	[ { "id": "34353", "date": "14 Jun 2018", "name": "Alexey Ruzov", "expertise": [ "Reviewer Expertise Epigenetics", "DNA methylation and demethylation" ], "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 manuscript by Germanguz et al. investigates potential roles of active DNA demethylation and the component of DNA base excision repair (BER) pathway, thymine DNA glycosylase (TDG) in cell cycle progression using human pluripotent stem cells (hPSCs) as a model. The authors first examined the expression levels of TDG in hPSCs, hPSC-derived neuronal progenitor cells (NPCs) as well as in the tissue-derived NPCs. Although the authors report a significant increase in expression of TDG in the hPSC-derived NPCs compared to the NPCs derived from tissues, it is unclear how comparable these two NPCs derivatives are and, therefore, the rationale behind this comparison is not fully clear. Interestingly, the authors find that expression of TET enzymes responsible for active DNA demethylation mimic TDG expression pattern in hPSCs and NPCs. Taking this into account together with the fact that cellular proliferation diminishes following birth in neural tissues, the authors sought to examine whether this pattern of TDG expression is linked to the cell cycle progression. To this end, the authors found that TDG levels increase during G0/G1 and decrease following entry to S and G2/M phases of the cell cycle. Next, the authors enquired whether the decrease in the differentiation potential of NPCs following TDG KD is due to prolonged proliferative status and show 34 cell cycle related genes amongst the genes differentially expressed upon the TDG knockdown. Finally, using a cell cycle reporter, FUCCI, the authors attempt to link the dynamics of TDG levels during cell cycle with active demethylation of the hPSC genome. Although, the authors show that TDG levels increase in G0/G1 cells and that 5fC and 5caC begin to accumulate in S phase, given the absence of what happens to the levels of 5fC and 5caC during cell cycle following TDG KD, it is not possible to conclude if TDG impacts the cell cycle dynamics of these modifications from the presented results.\n\nOverall, I think that this manuscript presents some novel and interesting data and the authors have a potential to develop this story much further. At the same time there is a number of issues with the current paper that should be fixed before it can be indexed, in my opinion.\nIn addition to the discrepancies mentioned above that need to be, at least explained in the text, there are following points that would be necessary to address:\nThe authors do not show how their findings on the involvement of TDG in cell cycle regulation impact differentiation of NPCs to either neurons or glia (This can be done by performing MAB-seq on the genes directly involved in neuronal/glial differentiation). It is difficult to understand why TDG depleted NPC have the same ratio of neuron to glial differentiation as the wild type ones (Figure 2E). This is especially strange since it is now well established that affecting the methylation status of differentiating NPCs either directly through modulation of DNMT proteins or by modulating the active DNA demethylation pathway via TDG/TETs, impacts the neuronal to glial differentiation lineage switch.\n\nIt would make sense to confirm at least the most important findings using an independent siRNA. Moreover, there is no catalogue number for the siRNA used in the manuscript.\n\nThe effects of TDG depletion on the cell cycle regulation shown in Figure 5 are rather mild. I understand that this can be explained by transient nature of the siRNA mediated depletion but the conclusion that “Taken together, it seems clear that TDG plays a role in human pluripotent stem cell cycle regulation” is an overstatement from my point of view.\n\nIt is unclear from the Methods section what type of microscopy (conventional or confocal) was used for the cell imaging.\n\nMultiple images of the cell nuclei should be shown in Figure 6 for each condition.\n\nThe nuclei are incorrectly called “nucleotides” in the Figure 6 legend.\n\nBoth the introduction and discussion are rather short and extremely superficial. Some of the references are cited incorrectly or in improper place. Furthermore, there is a large number of factual sentences in these sections that are lacking any references. Taking this into account, both Introduction and Discussion should be completely rewritten.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "38594", "date": "02 Nov 2018", "name": "Rahul Prasad", "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\n13/11/2018: This referee report has been updated from a Not Approved to an Approved with Reservations, and an additional sentence added, to reflect additional feedback from the referees after their report was published.\nGermanguz et al. investigate the role of TDG in the differentiation and proliferation of human neural progenitor cells using RNA interference, RT-PCR, bisulfite sequencing, and cell cycle profiling with the FUCCI system. They show that TDG knockdown, using siRNA and assessed by immunofluorescence and supported by increased levels of 5caC and 5fC but not 5hmC, correlates with an overall decrease in proportion of differentiated cells (both neurons and glia) following induction, in contrast to the authors’ expectation of increased developmental maturity following TDG knockdown. RT-PCR data show expression of neural progenitor cell markers to be unaffected by TDG knockdown. The authors then investigate cell cycle changes using FACS following TDG knockdown and report an increased fraction of G2/M cells. The FUCCI cell cycle reporter system was used to show that TDG levels are decreased outside of G1. Methylation-assisted bisulfite sequencing (MAB-seq) was used to show evidence of changes in methylation intermediate modification levels in a putative promoter region of EGR1. The authors conclude that the reduction of TDG expression induces cell cycle specific changes that result in increased proliferation and reduced differentiation of neural progenitor cells. Overall the study uses a strong combination of experimental methods to show interesting findings but is likely a few experiments short of adding to the growing narrative involving TDG, demethylation, and cellular differentiation.\nOne point the authors do not account for is the potential for reprogramming of neural progenitor cells to different, undifferentiated progenitors. DNMT3B knockdown has been shown to cause an increase in neural crest cell markers, hypomethylation at the Sox 10 promoter region, and ultimately prolonged neural crest emigration1. Therefore it seems important to confirm that the neural progenitor cells that failed to fully differentiate remained in the neural cell lineage instead of reprogramming to neural crest precursors – if reprogramming did occur this would be a significant finding.\nThe cell cycle data are not clear; there seems to be a discrepancy between the marked effect of TDG knockdown as assessed by Ki67 staining (Fig. 5A), which suggests an increased fraction of proliferating cells, and the rather subtle effect as assessed by FACS (Fig. 5B). In particular, the authors should show the percent of cells in G1, S and G2M by FACS in control and TDG knockdown. Also, the authors should clarify why the overall DNA content appears to be reduced in TDG knockdown cells.\nThe reported increased fraction of G2/M cells following TDG knockdown using FACS merits further investigation. The use of propidium iodide prevents noting a distinction between increased proliferation (increased mitotic cells) or if the cells are simply arrested in G2. This can be resolved by using DAPI with a mitotic marker such as phospho-histone H3.\nAdditionally, the authors do not investigate the effects of TDG knockdown using the FUCCI reporter system. This seems to be a technically feasible experiment that would serve as an important point in the study’s overall narrative linking TDG effects to the cell cycle.\nThe use of EGR1 as an example of methylation impacting gene expression is a point of concern. Selection of EGR1 as a gene of interest appears to be based on a single reference demonstrating methylation dependent expression in a rat model. Selection of a gene with a more established methylation dependent promotor region, such as Pax 6, to compare with the expression profile would be a better indicator of whether TDG knockdown affects differentiation2. We would argue that the authors should show changes in methylation intermediate levels by TDG knockdown, using MAB-seq, in more than a single gene. A minimum of three genes would seem to be appropriate to convincingly show an effect of TDG on gene expression via changes in methylation intermediates.\n\nThe following points would be helpful to be addressed:\n\nThe reduction of 5hmC levels in early S phase, as demonstrated in figure 6B is mentioned as previously reported but without citation in the main text.\n\nIn Figure 6B, 5hmC levels are shown to reduce in early S relative to G1, consistent with the coinciding increase in 5fC and 5caC levels. However, while 5fC and 5caC levels remain elevated in S, G2, and M compared to G1, 5hmC levels return to G1 levels and statistically higher levels than early S phase. This is a paradoxical finding and should be addressed.\n\nIn the methodology described for MAB-seq, the use of phenol/chloroform extraction is noted. This method has an established risk of sensitizing DNA to oxidation and should be addressed3, and therefore artifactually increase the levels of the oxidized cytosine species 5fC and 5caC. It is recommended that alternative methods of extracting DNA are used after each M.SssI treatment, such as Agencourt AMPure XP Beads4.\n\nMinor points: Normalized is mispelled in the figure legends. Several references are missing in the Introduction and throughout the paper, such as: Cortellino et al. (2011) on TDG knockout and embryonic lethality. Hardeland et al (2007), Slenn et al. (2014) and Shibata et al. (2014), on the decrease of TDG levels in S-phase. In the Introduction, what are the references for the sentence: “Loss of methylation in specific locations…”? In Fig. 2E what are the p-values for the differentiation? In particular, is the difference in GFAP-positive cells between control and TDG-KD significant? In the MAB-seq results, can the authors clarify the sentence: “This locus was chosen as it was reported (which reference?) to have the highest distribution of 5fC, 5caC around the TSS”? For the MAB-seq analysis in Fig. 4, it would be useful to have a schematic map showing the relationship of the CpG island to the TSS. In the paragraph on the FUCCI results, “DNA glycosylation activity of TDG” should be changed to “DNA glycosylase activity of TDG”. In the paragraph on the FUCCI results, it is not clear whether the FUCCI system was employed in hESC or hPSC cells. In the paragraph on the FUCCI results, the last sentence: “Furthermore, all these data…” is not clear and should be rewritten. The Discussion in the present form is a bit unclear, and should be rewritten, e.g. the last sentence is particularly imprecise. Also, references should be added to the Discussion. In Fig. 4C, change “5C” to “C”.\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/7-497
https://f1000research.com/articles/7-496/v1	26 Apr 18	{ "type": "Research Article", "title": "Social capital associated with quality of life among late adults and elderly population in the Northeast of Thailand", "authors": [ "Kitti Prachuntasen", "Wongsa Laohasiriwong", "Amornrat Luenam", "Kitti Prachuntasen", "Amornrat Luenam" ], "abstract": "Background: Previous studies indicated that social capital (SC) has an influence on quality of life (QOL). However, there are limited studies on how SC might associate with QOL among late adults and elderly in Thailand. Methods: This cross-sectional study was conducted among 1,148 participants who were identified by multistage random sampling from 4 provinces in the Northeast of Thailand. A self – administered questionnaire was developed and used to assess cognitive social capital (CSC), structural social capital (SSC), accessibility to health services, and socioeconomic status (SES) and QOL. The Generalized Linear Mixed Model (GLMM) was used to determine the association between SC and QOL when controlling for other covariates. Results: Only 41.03% (95%CI: 38.17 to 43.94) of the participants had good QOL. About half (50.26%) had high level of CSC, whereas only 36.15% had high level of SSC. The multivariate analysis indicated that having high levels of CSC and SSC was associated with good QOL. Other factors that were associated with having good QOL were aged <60 years old, monthly income ≥15,000 baht, adequate income, adequate physical activity, lived in the municipality, and had high level of accessibility to health services. Conclusion: Less than half of late adults and elderly had good QOL and high level of SSC. About half had high level of CSC. Both CSC and SSC had influence on QOL as well as gender, age, monthly income, financial status, physical activity, residential area, and accessibility to health services.", "keywords": [ "Social Capital", "Quality of Life", "Late Adults and Elderly Population", "Thailand" ], "content": "Introduction\n\nQuality of life (QOL) is the extent to which personal perception on comfortable or satisfying of their life in relation to their objectives, expectancy, standards, and concerns. It involves health and happiness, rather than wealth1,2. It includes all interpersonal and social capital (SC)3.\n\nSC is considered an important imperceptible health resource4. With growing recognition of the socioeconomic status (SES), and the social determinants of health, SC is now considered more significant to building healthcare systems5–7. SC has been described as a feature of social organization, for instance, norms, reliance and networks that can enhance the performance of community by accommodate coordinated actions8. The measuring of SC can maybe provision worthy insights into social networks and also connections that persons and communities have and more importantly, how these networks and connections possible have been utilized to encourage the positive outcomes for the person and the community. In this manner, the measuring of SC might augment our understanding of how persons in the community can work cooperatively to manage their problems and to accomplish their common goals. In addition, previous studies indicated that SC was associated with QOL. Particularly Majid Karimzadeh et al. (2013)9 indicated that SC is considered as a significant determinant of QOL. This SC is the sum of the actual or potential resources which are linked to a durable network of institutionalized relationships of generalized trustworthiness and active involvement that lead to mutual acquaintance or recognition, which are made up of social obligations or connections. The SC could convertible into economic capital and may be institutionalized in the form of a title of nobility10. Accessibility to health services can be highly influential on QOL and is related to both health care utilization and health outcomes such as stage at diagnosis and mortality11.\n\nThe Northeast of Thailand, the biggest region both in terms of areas and containing one- third of Thailand's 68 million populations. It is the poorest region in the country. Many of Northeastern migrate to work in other regions and overseas. Many of them usually leave family members include elderly at home. This socioeconomic situation might impact people way of life and their SC. The SC is importance in the culture in which they live. The National Bureau of Statistics indicated that SC matters definitely generate impact on QOL in the Northeast of Thailand, demonstrating results that will be applicable to other developing countries in the future.\n\nThere have been limited studies on SC among the Northeasterners and its association with QOL. Hence, the aims of this study were to investigate the prevalence and association between the SC and QOL in the Northeast of Thailand. The evidence from this study could be used as inputs for relevant sectors to develop measures in order to improve health and reduce inequalities in health through healthy public policy and strengthening community actions.\n\n\nMethods\n\nThis cross-sectional study was conducted during June 2016 to September 2017 in the 4 provinces of the Northeast region of Thailand include Loei, Khon Kean, Ubon Ratchathani and Nakhon Ratchasima provinces.\n\nThe inclusion criteria for participants were those who aged 40 years old or more, have lived in the Northeast of Thailand, could verbally communicate with the researchers and agree to participate with written inform consent. The exclusion criteria were severe impairment and/or having mental illness which limited their communication with the researchers.\n\nThe multiple logistic regressions used to identify the associations between multiple independent variables and a dichotomous outcome of QOL being high12 was used to estimate the sample size. The approximate sample size was 218. In order to control the over-fitting, we used ρ and variance inflation factor (VIF)13 which were 0.9 and 5.26, respectively. Therefore, the total number of samples was 1,148.\n\nA multistage random sampling method was used to choose participants of 4 provinces in the Northeast of Thailand, include Loei, Khon Kean, Ubon Ratchathani and Nakhon Ratchasima provinces. Firstly 4 provinces were randomly selected, and then 2 districts of each province were randomly selected namely Wangsaphung, Chiang Khan, Ban Phai, Chum Phae, Mueang Ubon Ratchathani, Det Udom, Phimai, and Sikhio, respectively. Third, three sub-districts from each province were randomly selected namely Pha Bing , Pak Puan, Khok Khamin, That, Chiang Khan, Na Sao, Mueang Phia, Hin Tang, Ban Phai, Wang Hin Lat, Non Sa-at, Nong Phai, Pathum, Rai Noi, Nong Bo, Mueang Det, Som Sa-at, Kaeng, Rang Ka Yai, Nong Rawiang, Bot, Wang Hin Lat, Non Sa-at, Nong Phai, respectively. Therefore 24 sub-districts were included in the study. The fourth stage was random selection of a village in each the sub-district. Then simple random sampling was applied to select 1,148 individuals proportionate to the size of the population to be included in this study. All participants were interviewed face-to-face by trained interviewers. The interview were performed door to door.\n\n\nResearch tools\n\nThe research tool was a structured questionnaire which was developed from reviewing literature. The questionnaire underwent content validation by 3 experts and then it was revised to improve validity. The questionnaire was tested for reliability using Conbach alpha amongst 30 participants who had similar characteristics with the samples in Nakhon Phanom province. Its Conbach alpha coefficient was 0.96. The questionnaire consisted of four parts: (1) socioeconomic (2) social capital; (3) accessibility to health services; and (4) quality of life (Supplementary File 1).\n\n\nAssessment of quality of life\n\nQOL was assessed using the Quality of Life scale (WHOQOL – BREF) of WHO Thai short version14. It is consisted of 26 items within the 4 domains containing physical, psychological, social relationships and environment domains. The scores are categorized into 3 groups: poor level (26–60 scores), fair level (61-95 scores) and good level (96–130 scores). Since the multiple logistic regression analysis of Hsieh is for the dichotomous outcome13. Therefore, in the bivariable and multivariable analysis we dichotomized the QOL as good QOL and poor/fair QOL, using cutoff point of below 96 for poor/fair QOL and 96 or above for good QOL15.\n\n\nSocial capital measurement\n\nThis study applied the concept of cognition and structure SC developed by Bain and Hicks in 1998. Six dimensions of cognition social capital (CSC) were examined in this study consisted of trust, solidarity, reciprocity, attitudes, behavior and social norms. Four dimensions of structure social capital (SSC) were included: horizontal organizational structure, accountability of leaders, collective/transparent decision-making process and practices of collective action and responsibility16. The response ranged from strongly disagree (1) to strongly agree (5). The SC scores are between 50 and 250. The higher scores reflected greater SC. Since the multiple logistic regression analysis of Hsieh is for the dichotomous outcome13. Therefore, in the bivariable and multivariable analysis the scores of cognitive social capital and structural social capital were dichotomized, using the mean as the cutoff point, as low/moderate (<3.68 scores) and high (≥3.68 scores),\n\n\nAccessibility measurement\n\nThe concept of access to health services of Penschansky & Thomas, 198117 was utilized in this study. The accessibility to health services was determined in term of individual’s satisfaction on health services covering the domains of accessibility, availability, affordability accommodation and acceptability. The scores ranged from strongly disagree to strongly agree (1 to 5 scores). The possible scores ranged 35–175. Higher scores mean higher accessibility. In the associations analysis the mean scores of accessibility to health services were dichotomized since the multiple logistic regression analysis of Hsieh is for the dichotomous outcome13, as low/ moderate (<3.68 scores) and high (≥3.68 scores).\n\n\nSocio-economic status (SES) and risk factors\n\nSocio-economic status in this study composed of gender, age, weight, high, educational attainment, marital status, occupation, monthly income, financial status, smoking, alcohol consumption, physical activity, residential area, and chronic diseases were treated as covariates in the analysis.\n\nTo minimized information bias, we trained interviewers and standardized the data collection competency in Loei, Khon Kean, Ubon Ratchathani and Nakhon Ratchasima provinces at our interviewer training workshop organized in Khon Kaen province. After the participants understanding of the purpose of this study and then signed consent informs. They were structured questionnaire interviewed by well-trained and standardized interviewers.\n\n\nData analysis\n\nSTATA® (ver. 13; College Station, TX, USA: Stata Corp). The categorical data were presents as frequency and percentage whereas the continuous data were explained their magnitude as mean, standard deviation, median, and range.\n\nThe GLMM was operated to model the random effects and correlations inside clusters18 since our study used multistage random sampling methods to choose the participants. In the modeling, the participant residential area was fixed whereas the aged was set as the random effect. Classification of QOL was done using univariable analysis, displayed as percentage of good QOL and its 95% confidence interval (CI).\n\nBivariable analysis was utilized to define the association of each independent variable with QOL. Any independent variables, in the bivariate analysis that had p ≤0.25 were chosen and continue to the multivariable analysis13.\n\nIn order to control the random effects and correlations inside clusters, the GLMM were operated before using multiple logistic regressions to determine the association between social capitals, residential area, accessibility to health services, gender, age, monthly income, financial status, and physical activity and QOL while controlling covariates (BMI, marital status, education, smoking, alcohol consumption, chronic disease, and occupation). The final model results presented the magnitude of association of independent variables and QOL were adjusted odds ratio (adjusted OR), and 95% CI.\n\n\nEthics statement\n\nThis research proposal and tool got approval from the Khon Kaen University Ethics Committee in Human Research, the approval number, HE 602127). Written informed consent was gathered from all participants.\n\n\nResults\n\nThe characteristics and SES of the participants are shown in Table 1. The majority of participants were female (60.63%), married (78.05%), completed elementary school (58.01%), with the average age of 58.36±11.95 years old, live in rural areas (53.48%). As high as 41.81% had average monthly income 5,000 – 15,000, 46.34% were unemployed, 47.3% had adequate income but not saving. Majority were overweight and obesity 54.35%. Most of them did not drink alcohol (75.70%), were not smoking (81.71%), had adequate physical activity (67.86%) Almost one third had chronic disease (32.06%). About half of participants had high cognitive social capital (CSC) (50.26%). Only 36.15% had high level of SSC. About one third (35.28 %) had low level of accessibility to health care services.\n\nMost of the participant (58.19%) had fair level of QOL followed by good (41.03%) and poor levels (0.78%) (Table 2).\n\nThe bivariate analysis revealed that both cognitive and structural social capital had influences on QOL. Other factors including gender, age, education, marital status, education, monthly income, occupation, financial status, physical activity, residential area, and accessibility to health care services also associated with QOL. The factors with p<0.25 in the bivariable analysis were proceed to the multivariate analysis (Table 3).\n\nMGLMM was performed to control the clustering effect of the sampling method in selecting the participants. The association between multiple independent variables and QOL was determine using multivariate analysis to control the effect of covariates. The results indicated that high levels of CSC (adj. OR = 2.83; 95%CI: 2.01 to 3.97) and high levels of SSC (adj. OR = 2.64; 95%CI: 1.84 to 3.76) were associated with QOL. Other demographic and socioeconomic factors that were also associated with good QOL were being male (adj. OR = 1.71; 95%CI: 1.27 to 2.31), aged <60 years old (adj. OR = 2.70; 95%CI: 1.72 to 4.24), monthly income ≥15,000 baht (adj. OR = 2.82; 95%CI: 1.89 to 4.23), had adequate income (adj. OR = 2.14; 95%CI: 1.54 to 2.97), lived in the town municipality (adj. OR = 2.52; 95%CI: 1.32 to 4.80). Others associated factors were had adequate physical activity (adj. OR = 1.54; 95%CI: 1.13 to 2.11) was associated with good QOL and had high levels of accessibility to health services (adj. OR = 1.53; 95%CI: 1.10 to 2.12). The results are shown in Table 4.\n\n* Adjusted for the effects of BMI, marital status, education, smoking, alcohol consumption, chronic disease, and occupation\n\n\nDiscussion\n\nThe main finding was discussed according to the objective of the study as follows: QOL measured by the WHOQOL – BREF. Majority of participants (58. 19%) had fair level QOL, a followed by good level (41.03%). This finding is similar to evidence indicated in Somrongthong R et al.19 that the majority of participants (58.19%) were at the “fair level”. These two studies were similar in the way that they were conducted in the Northeast. However, they might share similar social economics factors. However, it was lower than study by Hongthong D et al20. Which found that most of the samples (68.5%) had fair level of QOL. This study was conducted among rural settings which might contribute to rather lower level of QOL. About half of participants had high level of CSC and about one third had high level of SSC. It was confirmed by the results of the multivariable analysis that there was significantly association between SSC, CSC and QOL. The participants with high levels of SSC were 2.64 (95% CI: 1.84-3.76) times higher odds of having good QOL. It is possibly because people who participate in political, sports and occupation association or groups may lead to high SSC in term of scope and intenseness of links both within and outside the community. The more social connectedness, political participation, civic engagement they had the more satisfaction with life and relationships were found.\n\nThose with high level of CSC had 2.83 (95%CI: 2.01 to 3.97) times higher QOL than those with lower CSC. A possible explanation for this finding could be the CSC indicates capability of people to obtain various resources of social particular from their family, community, society, and health sectors21. People with high CSC are more likely to enthusiastic to find out for information, spiritual contribute networks. They tend to perform with social norms and peer control, could establish trust and be alive with others22–26.\n\nConcerning accessibility to health services, this study indicated that about one-third of participants had chronic diseases within the past year (32.06%). The participants who had better access to health services were 1.53% (95%CI: 1.10-2.12) higher odds of having high QOL. Access health services have intense effect on every aspect of quality of life. Access to quality health services is significant for promoting health, preventing disease, reducing disability and mortality which is a component of QOL.\n\nResidential area also influenced people’s QOL. In residential area, the influence of different lifestyles and stages of life have to be taken into account. The findings of this studies indicated that participant who lived in the town municipality had high QOL. This may be because the town municipality has good standard of living, good environmental health, better access to goods and health services, and better economic conditions27.\n\nOur result noted that gender was associated with QOL, of which similar with the evidence found in other studies that QOL was significantly varied with gender28,29. This study has shown that females had significantly poorer QOL when compared with males. Globally, some studied also indicated that females had lower QOL than males30–32. It was found that 60.72% and 61.41% of females who reported having lower education and monthly income, respectively than males, of had lower QOL. In some studies, higher education and income participants usually had higher QOL scores30,33. It is common in some countries that females were less privileged with inaccessibility to information and education resulting in disadvantages for employment, economic status and social position. Even though, women lived longer than men, they were more likely to suffer ill health than men34.\n\nThe QOL was significantly lower among participant among older persons30. Moreover, with increasing age, the QOL were decreasing, which was consistent with previous studies showed that elderly individuals suffering from poor QOL35. This study provides further evidence the older the individual, the QOL decreases. As the age advances, the health related problems become more common to a person. Gradually people are more weakening and finally confined to their own houses. Loneliness is commonly present. As age increases, the chance of losing spouse becomes more, loneliness increases to a much greater extent. Therefore, overall the physical and psychological QOL becomes poorer with the advancement of age. Beside this, the social interaction decreases with increased age. So, the social relationship domain of QOL becomes worse36.\n\nThis study also indicated that monthly income was associated with QOL. Those who had lower monthly income had lower level of QOL33. A possible explanation for this finding could be that poverty causes poor health status30. Different in employment status and occupation resulted in different income. Therefore, they would have different social security and health insurance which have influence on their health and wellbeing32. The increase in per capita income indicates better socio-economic status36. An increase in per capita monthly income significantly improved the QOL scores.\n\nA profound association was found between having adequate income and QOL. Participants who were poor with inadequate income faced various problems. Having sufficient income is important to QOL It help covering, meeting the basic needs of life. In addition it also increases the opportunity to participate in social activities to enjoy hobbies, holidays and luxuries37.\n\nPhysical activity also had high influence on QOL and has been profoundly discussed in the literature38. In this study active physical activity was associated with QOL. For instance, good QOL was found 1.54 times more among participants who had adequate practiced physical activity.\n\nThis study has indicated both the QOL and social capital situations and their association which are highly useful to planning for relevant actions. However, it is important to aware that a limitation of cross-sectional study is that because the exposure and outcome are simultaneously assessed, no causal relationship can be identified. It is important suggestion for a further study which could identify the causal relationship using other study designs such as a cohort study.\n\n\nConclusion\n\nThis community based cross sectional found that less than half of late adults and elderly had good QOL and high level of SSC. About half had high level of CSC. Both CSC and SSC had influence on QOL as well as gender, age, monthly income, financial status, physical activity, residential area, and accessibility to health services. Relevant sectors could use the evident from this study to develop proactive strategies and measures to improve health and reducing inequalities in health through healthy public policy and strengthen community actions to improve their social capital, access to health care and socioeconomic status.\n\n\nData availability\n\nDataset 1: Social capital associated with quality of life among late adults and elderly population in the Northeast of Thailand. Zip file containing raw data and word document with coding scheme for the dataset. 10.5256/f1000research.13954.d20141439", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was financially supported by the Research and Training Center for Enhancing Quality of Life for Working Age People, Khon Kaen University, Thailand.\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 express sincere thanks and appreciation to all participants who participated in this study.\n\n\nSupplementary material\n\nSupplementary File 1 – Questionnaire used in the study.\n\nClick here to access the data.\n\n\nReferences\n\nSkevington SM, Lotfy M, O'Connell KA: The World Health Organization's WHOQOL-BREF quality of life assessment: psychometric properties and results of the international field trial. A report from the WHOQOL group. Qual Life Res. 2004; 13(2): 299–310. PubMed Abstract \| Publisher Full Text\n\nWHOQOL Group: Study protocol for the World Health Organization project to develop a Quality of Life assessment instrument (WHOQOL). Qual Life Res. 1993; 2(2): 153–9. PubMed Abstract \| Publisher Full Text\n\nBonomi AE, Patrick DL, Bushnell DM, et al.: Validation of the United States' version of the World Health Organization Quality of Life (WHOQOL) instrument. J Clin Epidemiol. 2000; 53(1): 1–12. PubMed Abstract \| Publisher Full Text\n\nHsieh CH, editor: A concept analysis of social capital within a health context. Nurs Forum. Wiley Online Library. 2008; 43(3): 151–9. PubMed Abstract \| Publisher Full Text\n\nMurayama H, Fujiwara Y, Kawachi I: Social capital and health: a review of prospective multilevel studies. J Epidemiol. 2012; 22(3): 179–87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStory WT: Social capital and health in the least developed countries: a critical review of the literature and implications for a future research agenda. Glob Public Health. 2013; 8(9): 983–99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFujisawa Y, Hamano T, Takegawa S: Social capital and perceived health in Japan: an ecological and multilevel analysis. Soc Sci Med. 2009; 69(4): 500–5. PubMed Abstract \| Publisher Full Text\n\nNyqvist F, Forsman AK, Giuntoli G, et al.: Social capital as a resource for mental well-being in older people: a systematic review. Aging Ment Health. 2013; 17(4): 394–410. PubMed Abstract \| Publisher Full Text\n\nKarimzadeh M: Impact of social capital on quality of life: Evidence from India. IJEPT. 2013; 3(4): 264–71. Reference Source\n\nBourdieu P, Wacquant LJ: An invitation to reflexive sociology. University of Chicago press; 1992. Reference Source\n\nMaliski SL, Connor SE, Oduro C, et al.: Access to health care and quality of life for underserved men with prostate cancer. Semin Oncol Nurs. 2011; 27(4): 267–77. PubMed Abstract \| Publisher Full Text\n\nCohen N, Galea S: Population mental health: Evidence, policy, and public health practice. T&F. 2011. Reference Source\n\nHsieh FY, Bloch DA, Larsen MD: A simple method of sample size calculation for linear and logistic regression. Stat Med. 1998; 17(14): 1623–34. PubMed Abstract \| Publisher Full Text\n\nMahatnirundkul S: Comparison of the WHOQOL-100 and the WHOQOL-BREF (26 items). J Ment Health Thai. 1998; 5: 4–15. Reference Source\n\nTaboonpong S, Suttharangsee W, Chailangka P: Evaluating psychometric properties of WHO quality of life questionnaire in Thai elderly. J Gerontol Geriatric Med. 2001; 2: 6–12.\n\nBain K, Hicks N: Building social capital and reaching out to excluded groups: the challenge of partnerships. World Bank. 1998.\n\nPenchansky R, Thomas JW: The concept of access: definition and relationship to consumer satisfaction. Med Care. 1981; 19(2): 127–40. PubMed Abstract\n\nHubbard AE, Ahern J, Fleischer NL, et al.: To GEE or not to GEE: comparing population average and mixed models for estimating the associations between neighborhood risk factors and health. Epidemiology. 2010; 21(4): 467–74. PubMed Abstract \| Publisher Full Text\n\nSomrongthon R, Wongchalee S, Yodmai K, et al.: Quality of Life and health status among Thai elderly after economic crisi, Khon Kanen province, Thailand. Eur J Sci Res. 2013; 112(3): 314–24.\n\nHongthong D, Somrongthong R, Ward P: Factors Influencing the Quality of Life (Qol) Among Thai Older People in a Rural Area of Thailand. Iran J Public Health. 2015; 44(4): 479–85. PubMed Abstract \| Free Full Text\n\nHu F, Niu L, Chen R, et al.: The association between social capital and quality of life among type 2 diabetes patients in Anhui province, China: a cross-sectional study. BMC public health. 2015; 15(1): 786. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLucumi DI, Gomez LF, Brownson RC, et al.: Social capital, socioeconomic status, and health-related quality of life among older adults in Bogotá (Colombia). J Aging Health. 2015; 27(4): 730–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeshmukh PR, Dongre AR, Rajendran K, et al.: Role of social, cultural and economic capitals in perceived quality of life among old age people in kerala, india. Indian J Palliat Care. 2015; 21(1): 39–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim D, Kawachi I: U.S. state-level social capital and health-related quality of life: multilevel evidence of main, mediating, and modifying effects. Ann Epidemiol. 2007; 17(4): 258–69. PubMed Abstract \| Publisher Full Text\n\nRimaz S, Mohammad K, Dastoorpoor M, et al.: Investigation of relationship between social capital and quality of life in multiple sclerosis patients. Glob J Health Sci. 2014; 6(6): 261–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKleinman A, Wang WZ, Li SC, et al.: The social course of epilepsy: chronic illness as social experience in interior China. Soc Sci Med. 1995; 40(10): 1319–30. PubMed Abstract \| Publisher Full Text\n\nSahin NP, Fasli M, Vehbi BO: An assessment of quality of life in residential environment: case of Selimiye Quarter in Walled City of Nicosia, north cyprus. 2007; 23: 2012. Reference Source\n\nBurström K, Johannesson M, Diderichsen F: Health-related quality of life by disease and socio-economic group in the general population in Sweden. Health Policy. 2001; 55(1): 51–69. PubMed Abstract \| Publisher Full Text\n\nScalone L, Cortesi PA, Ciampichini R, et al.: Health related quality of life norm data of the Italian general population: results using the EQ-5D-3L and EQ-5D-5L instruments. Epidemiol Biostat Public Health. 2015; 12(3). Reference Source\n\nShiroiwa T, Fukuda T, Ikeda S, et al.: Japanese population norms for preference-based measures: EQ-5D-3L, EQ-5D-5L, and SF-6D. Qual Life Res. 2016; 25(3): 707–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerreira LN, Ferreira PL, Pereira LN, et al.: EQ-5D Portuguese population norms. Qual Life Res. 2014; 23(2): 425–30. PubMed Abstract \| Publisher Full Text\n\nSun S, Chen J, Johannesson M, et al.: Population health status in China: EQ-5D results, by age, sex and socio-economic status, from the National Health Services Survey 2008. Qual Life Res. 2011; 20(3): 309–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo N, Johnson JA, Shaw JW, et al.: Self-reported health status of the general adult U.S. population as assessed by the EQ-5D and Health Utilities Index. Med Care. 2005; 43(11): 1078–86. PubMed Abstract \| Publisher Full Text\n\nAnnandale E, Hunt K: Gender inequalities in health: research at the crossroads. 2000. Reference Source\n\nTajvar M, Arab M, Montazeri A: Determinants of health-related quality of life in elderly in Tehran, Iran. BMC Public Health. 2008; 8: 323. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDatta D, Datta PP, Majumdar KK: Association of quality of life of urban elderly with socio-demographic factors. IJMEDPH. 2015; 5(4): 274–278. Publisher Full Text\n\nBowling A: Ageing well: Quality of life in old age: McGraw-Hill Education (UK).2005. Reference Source\n\nSampaio PY, Ito E: Activities with higher influence on quality of life in older adults in Japan. Occup Ther Int. 2013; 20(1): 1–10. PubMed Abstract \| Publisher Full Text\n\nPrachuntasen K, Laohasiriwong W, Luenam A: Dataset 1 in: Social Capital Associated with Quality of Life among Late Adults and Elderly Population in the Northeast of Thailand. F1000Research. 2018. Data Source" }	[ { "id": "33481", "date": "08 May 2018", "name": "Farid Najafi", "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 cross-study on association between social capital and quality of life in Thailand. The justification of study has been provided in \"Introduction\". However, there are many studies on such associations that authors can cite some of them in Introduction.\nWhile the details of multistage random sampling have been provided, justification for calculated number of participants (second paragraph under the subheading of \"study participants\") has been written in a wrong way and need to be re-written.\nWe did not know \"Conbach alpha\". It might be Cronbach's alpha. Justification for classification of SC as a dichotomized variable is not correct and sufficient. This is true for accessibility to health service.\nThe manuscript need severe English language editing service.\nThe presentation of Tables is understandable and appropriate.\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? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "37414", "date": "16 Jul 2019", "name": "Agnes Iok Fong Lam", "expertise": [ "Reviewer Expertise communication", "media", "public 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\nThis paper shows the correlation between good quality of life and social capital and some factors like gender, income, and accessibility to health services. It is an analysis of the situation of Northeast of Thailand that can inform policy needs for the community. However, it is worth to point out the following:\n\nThe “Introduction” is not comprehensive enough to understand the context, especially for readers outside Thailand. Census information on the population (the Northeast of Thailand, paragraph 2) should be more concise and specific for the denotation like “It is the poorest region in the country.” Also, it would be much more convincing if studies are provided to support statements like \"many of Northeastern migrate to work in other regions and overseas. Many of them usually leave family members to include the elderly at home.\" For status like \"there have been limited studies on SC among the Northeasterners and its association with QOL,\" it is expected that to quote a few (like one or two) of these studies and pointed out their relevance to the topic.\n\nThe conclusion is sound, and the figures are clearly stated and shown. However, discussion into the context will be necessary — for example, demographic statistics (Table 1. Characteristics of participants in the Northeast of Thailand) shows that the percentage of employed participants that work as \"Government servant\" is very high, 13.85% out of the total population, it is more than one-quarter of the employed population. In Table 4 (Multivariate analysis for factors associated with good quality of life (QOL) using GLMMT), employment status was not shown as a factor associated with good quality of life. Since \"Government servant\" is the second largest industry among the survey population, just after agriculture, it would be worth to add some observation over the employment issues even the cross-sectional data analysis shows that employment stats is not significant. Demographic data shows that the studied area has a unique market structure, and the paper needs to elaborate if the result could be applied to the rest of Thailand or some other middle-income developing 's countries.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-496
https://f1000research.com/articles/7-495/v1	26 Apr 18	{ "type": "Opinion Article", "title": "The need for a new science, technology and medicine University in the Middle East: Carving the future", "authors": [ "Zaheer-Ud-Din Babar" ], "abstract": "With innovations in science and technology, the world is transforming at a fast pace. The Universities in today’s world, their structures, differences, and the work they do could have a significant impact on the lives of the people. This opinion piece proposes that a new Science, Technology and Medicine University is needed in the Middle East. The article also argues why such a University is needed in the presence of other Universities. This proposed University could act as a global think tank and could bridge the ideas, gaps, and work between low, middle and high-income countries. Currently, in the Middle East (in Dubai), several large western Universities have opened their campuses. The initiatives like this would certainly improve the quality of education, however, this also indicates that there are gaps and the need to build the more educational institution. Specifically, an institution in the region is needed with a global outlook and approach; a centre or powerhouse of ideas. In this paper, I briefly discuss the proposed University, its people, scope, vision, geographical areas to cover, financial models and the stakeholders who might be interested to build this University.", "keywords": [ "Middle East", "Dubai", "Science", "Technology", "Medicine", "University", "Future", "Ideas" ], "content": "Introduction\n\nThough the Universities in the modern world differ from the past, however, the basic structures and functioning remain the same (University Alliance: Growing the future). In order to progress further, rather than being standalone academic institutions, Universities will need to become more integrated into the economy, with real commercial awareness and relationship management (The 2018 University: how ready is higher education to embrace the future). Currently, the majority of the Universities react to change, they work towards a solution when a problem is presented. However, it is argued that the Universities can take a more proactive role in charting a future for humanity and can become more effective partners in global development. This has been highlighted in various reports published by World Bank, UK’s Department for Business Innovation and Skills and by the Centre for Higher Education Research and Information at The Open University in UK. The development agenda could include working on specific challenges and goals, for example, solving HIV or global hunger or providing a business solution for a problem of global scale.\n\n\nVision\n\nA Science, Technology and Medicine University in the Middle East with preferably off-shoot campuses in other low and middle-income countries (South and South-East Asia, Africa). The University will be producing human resource who could tackle the science and technology challenges in 21st century.\n\nA University fostering collaboration between global South and North. An area in the Middle East (for example Dubai) could act as a global hub. The University can work closely with the international agencies, WHO, World Bank and UN.\n\nIn the Middle East (in UAE, Dubai, and Qatar), several large western Universities have opened their campuses. This is a good approach and would strengthen the quality of education. However, though these Universities are established institutions, their main mandate is to offer programmes and courses of their parent institute. The large majority of these universities are not meant to take up regional problems, local issues or global agenda. However, there are some exceptions such as Johns Hopkins University Programs in Qatar. This program was founded on the belief that social and behavior change communication (SBCC) is key to solve pressing health problems.\n\nIt is a known fact that the lack of think tanks and innovative Universities in the global South (developing world) have negatively impacted on leadership, generation of new ideas and bringing a positive change. Think tanks in many developing countries lack the financial and human resources to conduct in-depth economic analysis and carry out their tasks effectively (Endowments for Think Tanks in developing countries: What role for private foundations and official donors? High-level Seminar, OECD Headquarters, Paris, 28 April 2008). The Universities in developing world were supposed to play a pioneering role in addressing problems of poverty, social disorganization, low production, unemployment, hunger, illiteracy, diseases1, however, these objectives are not achieved in the real sense. On the other hand, the gap in science and technology between developed and developing countries is growing at an alarming pace necessitating the need for innovative models for education. In this context, the initiatives like this could have a real impact and can become an effective partner for global development.\n\nThe University can work at several levels. It can synthesize the knowledge available at a global scale and provide themes, action points and active solutions to the problems. The University can also chart ways for the new programmes and courses and rather than just following a traditional pathway of teaching and learning, this University can create an agenda of its own. Please see Figure 1 on the University’s work, its’ key stakeholders and partners. The work could include but not limited to on the issues such as future of humanity, ecology, the outbreak of diseases, developing new medicines, climate change, food safety, artificial intelligence, etc\n\nHere are the five core principles to build the foundation of the University:\n\n1. As a driver for change: changing thinking, practices, developing new models to improve education and science\n\n2. University with an impact: impact on society, lives of people, consumers, patients, systems\n\n3. Global niche, design, scope, and vision: Education and research with an impact, to launch programmes which are vital but not being covered by other mainstream Universities\n\n4. Bridging the gap between developed and developing countries\n\n5. Providing sustainable education\n\n\nGeographical areas to cover\n\nThe region, because of its global outlook and population could benefit from an independent Science, Technology and Medicine University. Dubai as its main hub, while opening off-shoot campuses in other countries (South Asia, and Africa). The reason to choose Dubai is its close proximity to large population centres in Asia (China, India) Africa and the Middle East. These are the areas where the majority of the world’s population live and the impact of any initiative of this nature would be immense. However, it does not mean that the similar initiatives are not needed for other regions, for example, Latin America, South America or for the Pacific.\n\n\nThe people and financial models: Call for action\n\nThe University can be managed by a multidisciplinary group with either a profit or a not for profit set-up. The University would be truly global in nature with a strong presence of expertise from low and middle-income countries. It could come up with a broad mix of people including professionals, entrepreneurs, and scientists.\n\nThere are different financial models which can be explored to build this University. This includes consulting and service models as well as expertise and capacity building in specific areas. Turning to investors and entrepreneurs, as well as to government and donor agencies could be some other options. The University can be built as, a public/private mix, or either as a not for profit company.\n\nAs institutions working on futuristic global issues are scant, developing such a University is vital. It would help provide innovative solutions relating to health, medicine, science, and technology as well as solving pressing problems and needs. The international agencies are in the best position to take this idea further.", "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\nMosha HJ: The role of African universities in national developments. Higher Education. 1986; 15(1–2): 113–134. Publisher Full Text" }	[ { "id": "33835", "date": "13 Jun 2018", "name": "Sammy Ohene", "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 author makes a valid point on the need for universities that seek to provide solutions for the specific problems of less developed countries.\n\nThe fact of a large gap in science and technoloy between North and South is noted as well as the need to look to the future in developing programmes for such a new university.\nThere is no doubt a university owned and manned mainly by citizens of low and middle level countries will better be able to focus on local problems.\nIt is not clear however how such a new type of institution will address the issues of scientific deficit or dò better in the field of artificial intelligence than traditional universities.\nThe potential collaborative role is particularly exciting.\nThe recommendations for financing need further clarification since this is crucial for the realisation and sustainability of the idea.\n\nIt would be helpful to provide evidence for the assertion that \"lack of think tanks and innovative Universities in the global South have negatively impacted on leadership, generation of new ideas and bringing positive change.\"\nIt is not clear also if asking the World bank and similar global institutions to take it up means they should come up with the money or only develop the concept further.\nOverall I think it is a paper worthy of attention by anyone keen on trying a new way of fostering development and equality.\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? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "33839", "date": "23 Aug 2018", "name": "George Perry", "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 outlines a clear and important vision of a university for the world located in the Middle East. The goals of stimulating development in the \"South\" with a \"South\" orientation rather than a North one is well presented.\nThe arguments could be strengthened with (1) more concrete numbers, such as the number of students leaving the Middle East for Europe and the U.S. for an education who do not return; (2) the viability of current Middle East institutions to transform, as it seems some are doing at this time; (3) the possibility of investment from the North or Middle East wealth to make this feasible.\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": [] } ]	1	https://f1000research.com/articles/7-495
https://f1000research.com/articles/5-1903/v1	02 Aug 16	{ "type": "Opinion Article", "title": "Personal values influencing career path in academic medicine", "authors": [ "Marissa Tsoi", "Braden D. Teitge", "Christopher R. Madan", "Louis H. Francescutti", "Marissa Tsoi", "Christopher R. Madan", "Louis H. Francescutti" ], "abstract": "To pursue research, education, and health policy in one’s career, broadly defined as academic medicine, is one of the most important decisions of a trainee doctor’s career. Despite this, there is scant literature on which factors influence trainees’ choices towards clinical work or academic research. As the MD/PhD is a relatively young training path compared to the traditional PhD (Doctor of Philosophy) and MD (Doctor of Medicine) programs, it prompts the question: at the crossroads of a career, what sways the individual to select an MD, PhD, or MD/PhD program? This is a valuable question to be answered for trainees who are considering multiple career paths, for educators who want to guide undifferentiated students, and for policy makers who develop and coordinate research programs. “Intellectual stimulation” is the most consistently identified personal value which draws trainees to academic medicine. Mentorship is linked strongly to success in the field. Conversely, long training periods, a lack of autonomy, and financial considerations are deterrents from a career in academic medicine. Insight into the decision-making process is provided by recent trainees in these respective fields, as well as experienced academic clinicians.", "keywords": [ "medical education", "academic medicine", "personal values", "MD/PhD", "Canadian medical education", "undergraduate medical education", "postgraduate medical education", "continuous medical education" ], "content": "What is academic medicine, where does it start, and who chooses it?\n\nAcademic medicine is broadly defined as “the discovery and development of basic principles, effective policies, and best practices that advance research and education in the health sciences, ultimately to improve the health and well-being of individuals and populations”1. The interest in academic medicine comes from the fundamental tenet of modern medicine based on discovery, research and innovation. Physicians that are active in research and innovation help to keep medical knowledge and clinical care on the cutting edge, constantly improving and ensuring that we deliver the best care for our patients. Healthcare policy makers too must be familiar with health policy research to guide their decision-making and academic activity.\n\nEncouraging the next generation of physicians to become active in research and health policy can only help the advancement of clinical care, from the bench to the bedside. Trainees may undertake academic medicine at several points in their career. Many obtain MSc or PhD degrees prior to enrolling in medical school; others enroll in a combined MD/PhD program or complete a PhD degree during residency training; others yet decide later, as independent physicians, to add an academic component to their practice. Many residencies incorporate a strong academic aspect, especially for those completing fellowships in a sub-specialty.\n\nWith respect to the germination of this interest, it is recognized that research interest often begins in residency and medical school, with early exposure to research in medical school fostering interest in it as a career2. However, many factors determining career path are at play even earlier on, before the start of a graduate program or acceptance into a medical school. By virtue of entrance criteria, a decision whether to enter an MD or graduate program likely occurs around the time of undergraduate degree completion. Furthermore, it is interesting to examine the ongoing decision to remain in a chosen career pathway, for research has shown that trainees’ interest in academic medicine wanes as they progress2.\n\nDue to the various routes to a career in academic medicine, it is difficult to define distinct decision points in time when a trainee chooses to pursue academic medicine. Many extrinsic factors which influence career choice may vary over the course of this journey; this makes trainees' personal values relevant, if regarded as a consistent factor. Is there a type of individual who chooses academic medicine, and what values guide this person to an MD, MD/PhD, or PhD degree?\n\n\nThe MD/PhD: a distinct avenue in academic medicine\n\nThe MD/PhD combined degree is a clear and relatively modern pathway in structured academic medicine. Recent reductions in Canadian MD/PhD funding cloud its future, but its historical rise has been promising. The first Canadian MD/PhD program began in 1984 at the University of Toronto, with a starting class of 2 students. It has since grown to 42 students and is the largest program of its kind in Canada3. As of 2014, combined MD/PhD programs are offered at most medical schools in Canada4. The number of students accepted into a given school's MD/PhD program each year varies between 1–10 across Canada, and interestingly the students are mostly male, in keeping with overall trends in academic medicine5.\n\nThe demand for clinicians with different expertise, such as education, health policy, or business has attracted trainees and educators to vastly grow the training programs that are now available. Trainees may now also pursue an MSc, MBA, or similar degree at nearly any point in their career. This allows physicians to combine clinical care with an interest in managerial skills, leadership, or virtually any interest complementary to medical practice. The expansion of these academic training pathways is changing the landscape of medical research and clinical care, prompting the question of which values guide trainees to choose academic careers, and what this means for the future of academic medicine.\n\n\nChoosing academic medicine: pros, cons, and trends\n\nThere is an array of literature on the current state of academic medicine in North America2. The most frequently cited disincentives are length of training, lower financial reward, and lack of autonomy6. The pressure to assume the \"triple threat\" mantle of clinical work, research, and education can also dissuade students from academia. Program length, fear of burnout, difficulty juggling work-life balance, and advanced age at completion are also reasons for dissatisfaction7. Of course, other factors such as debt and family influences also put pressure on the trainee to enter the workforce. Perhaps most concerning is that senior residents report less interest in research than junior residents over time7.\n\nAcademic training that comes in addition to a 3–4 year MD degree delays the trainee’s potential professional level salaries. Until 2015, the Canadian Institutes of Health Research (CIHR) provided $21,000 (CAD) of grant funding per annum for 6 years to MD/PhD candidates8. The average income 2 years after completing one’s postdoctoral degree in Canada is $65,0009. One can attempt to compare this to a second-year MD resident’s salary of roughly $60,000, with a further substantial increase upon attaining a staff position at the end of residency10. Moreover, a standard MD degree takes at least 2–3 fewer years than either a PhD or an MD/PhD, therefore reducing time spent as a student.\n\nGender imbalance is a noted trend in academic medicine, as the overwhelming majority of those entering academics are male, and males in academic medicine have a greater salary. The salary difference between male and female early-career physician-researchers despite adjustment for work hours, specialty, and academic rank is over $30,00011. This gender imbalance certainly needs to be addressed and rectified, both to encourage females to enter academic medicine, and reward those that do make this decision. There are no robust studies examining the reasons for this, but a few cohort studies have shown that more female trainees lose interest in research over time compared to males12 and that women in academia were less likely to be married than those in private practice13. A study with a female focus group found that perceived inflexibility in clinical pathways and decreased ability to balance competing roles were disincentives for academic medicine14.\n\nIncentives drawing a trainee towards academic medicine include a passion for research, early exposure to research, desire to become an educator, desire for clinical appointment, and strong mentorship2. Most of the literature stresses that the earlier a trainee is involved in research and the more involved they are, the more likely they are to pursue academic medicine.\n\nIn terms of predicting who will enter and succeed in academic medicine, the strongest correlation is with the completion of a research fellowship or a degree such as a Masters, PhD or MD/PhD15. A joint degree such as the MD/PhD is often associated with faculty and academic appointment15. In a retrospective analysis of nearly 2000 medical graduates in the United States (1997–2002), those with a MD/PhD were more likely to have a full-time faculty appointment with an odds ratio of 2.3316. Publication of research conducted in medical school and residency correlated with trainees choosing academic medicine careers, as did attending a \"research-intensive\" university17,18.\n\nCurrent trends in academic medicine include a shift of physician-scientists from laboratories to more clinical departments, and an increase in competency-based programs, which give researchers flexibility in combined programs such as the MD/PhD7. In undergraduate medical education, there are numerous early academic tracks such as the MD with Special Training in Research (STiR, at the University of Alberta) and Research in Medicine (RiM, at Dalhousie University). These are aimed at cultivating an early academic interest in medical students. At the postgraduate level, there is the Clinician Investigator Program (CIP, at the Royal College of Physicians and Surgeons of Canada) for residents in sub-specialties, as well as the Clinician Scholars Program, offered by various medical schools across Canada, to both specialty and family medicine trainees.\n\n\nValues and the perspectives of individual trainees\n\nBorges et al. identified three divisions of values that influence the learner’s decisions to pursue an academic career: the individual’s personal values, the values of groups with which one associates, and generational values6. The only personal value consistently identified across studies was \"intellectual stimulation\"19. All other personal values leading to an academic career remain poorly defined6.\n\nMeanwhile, Shea et al. identified characteristics of mentors and mentees that were deemed most important by a think tank of American physician-scientist programs. Personal attributes emerged at the top of both lists: for mentees the most crucial themes were passion, focus, ability to communicate clearly, desire, dedication, discipline, and resilience. For mentors the themes were prior mentoring successes, emotional intelligence, altruism, the will to promote independence, and optimism20.\n\nThe following narratives from medical trainees and practicing physician-researchers attempt to shed light on the individual's personal values and the thought processes which guide career decisions towards or away from academic medicine. The five interviews were conducted by either email or telephone correspondence, from January to May 2015. The interviews were conducted, transcribed, and summarized by a single interviewer. Questions were tailored to each participant, but a basis of core questions is shown in Table 2. The participants were selected from the authors’ network of contacts to sample perspectives at each stage of academic medicine. All interviewees had completed some or all of their training at Canadian universities. The respondents consented to publication of their opinions, but were anonymized with respect to university, name, and location.\n\n\nPart 1: Considerations on prioritizing medical practice over research: the MD student perspective\n\nThis trainee stated that he enjoyed medical research, and found it was helpful to balance this with clinical work. He also wanted to work in an academic center, and found that a research background helped to open doors in major centers. He wanted research to be a distinct but relatively minor piece in his career, and did not feel the need to do additional training. Mentorship was also key, both in getting him initially involved in research and helping him navigate the waters of academia. This MD wanted to continue his involvement with research, because he enjoyed research, the mentorship, and the career opportunities it afforded him.\n\nAccording to this trainee, the biggest draw to research right now is the competitive advantage it gives for academic career positions. In major teaching hospitals, many people now need some kind of research background, and the numerous fellowships are pushing more and more people into research. Keeping them involved however, is a bit more intensive, as we need to make sure that the infrastructure and grant support is there for our clinician researchers who are already very time constrained.\n\nThis trainee stated that undergraduate experiences in research inspired her to attain a Masters degree, especially in a gap between undergraduate completion and application to medical school. She later chose to pursue an MD degree as she did not envision scientific research forming the major component of her career. The student was satisfied with continued research involvement while completing her medical degree. She had confidence at this stage with her research skills as a Masters graduate. Though open to consideration of a PhD in the future, she cited length of training and concerns over a possibly diminished quality of both clinical education and research as deterrents. In particular there was a concern due to the format of an MD/PhD program, wherein gaps between the respective phases might lead to loss of clinical skills or, on the other hand, less novelty of one’s research by training’s end. This trainee personally preferred a singular focus on clinical practice as opposed to splitting her attention between that and academic medicine.\n\n\nPart 2: What an MD adds or takes away from PhD training: the PhD student perspective\n\nThis trainee stated that prior to beginning his undergraduate program, he had planned to pursue an MD. As a means to gain useful experience as an undergraduate, he became involved in a research lab. However, through his undergraduate course work, he found that he did not enjoy some activities that he thought would be critical to being successful in medical school, memorizing anatomical nomenclature and dissection labs. Concurrently, he did find that he took well to research and was able to readily follow with the logical thinking and creative problem solving that are fundamental to academic research. This PhD student has since continued his research career as a postdoctoral research fellow, and plans to work towards a professor position at a research-intensive university. He commented that not having an MD and its related training have posed some limitations to his work, as he can not readily recruit patient populations without first finding an MD with relevant background and interests to collaborate with.\n\nWhile he does not think that everyone interested in obtaining an MD would be better suited with a MD/PhD, he does think that scientific literacy is an important skill for MDs, and that becoming involved in a research laboratory at the undergraduate level should be encouraged for students considering a medical career, even if just in volunteer capacity. Exposure to academia may make future doctors more readily able to incorporate scientific advances into their practice.\n\n\nPart 3: Balancing practice and research: the MD/PhD student perspective\n\nThis student chose the MD/PhD path because of the inspiring early undergraduate research experience she was involved with. These experiences ranged from volunteering in a laboratory doing benchwork, to interacting with patients in clinic. When she had a gap between undergraduate completion and application to medical school, her strong MD/PhD mentors, including the Program Director, guided her towards academic medicine.\n\nIn terms of personal values, this trainee had a strong sense of social justice and felt that her research was emotionally as well as intellectually stimulating. She felt that her attempts to effect large-scale change would be eased by having the MD/PhD degree. This was a way for her opinions to carry more weight, which would strengthen her ability to advocate for her patient population. As someone who was planning to spend half her future career in research and half in clinical duties, she was overall very well suited to her program.\n\nRegarding the gender divide, she noted that although numbers were equal for male and female trainees early in the program she found fewer female mentors on the tenure track and at academic conferences. Another personal value in her decision was her relatively young age at entry to the program. She felt that the MD/PhD path gave her more time to decide what field of medicine to devote herself to, and this was time she could afford.\n\n\nPart 4: Practicing MD returns to pursue a PhD: the lifelong-learner perspective\n\nThis experienced physician felt that his practice situation, at a community health centre, had stabilized over the years thanks to a team of seasoned colleagues. Now was a good time to ease up on clinical commitments and pursue a PhD in philosophy. From a personal perspective, he reflected on returning to academia having something to do with his time of life. He wished to continue intellectual challenges, broadening his understanding and knowledge, as well as analytic and argument skills. As he serves a low socioeconomic status population, he felt he could improve his value as a resource with this formal training in philosophy – by advocating more effectively. He cited his belief that we need to ask ourselves honestly what we owe one another in this world, a broad question that could not be answered within the narrow confines of medical practice. He envisioned teaching, mentoring and modeling a more pervasive and broadly-informed understanding of philosophy, morals and values as applied to the healthcare and other systems. He wished to place particular emphasis on a more complex and complete understanding of marginalized populations, and of the role of the physician in society. In seeing how society treats its marginalized sections, as well as many years of coming up against healthcare’s attitudes toward disadvantaged populations, he was convinced of the necessity to ask questions in a different way. A PhD in philosophy would help this physician formulate those questions to be more clear, meaningful, and effective.\n\n\nDiscussion\n\nWhile these are only a handful of individuals’ perspectives, in aggregate they provide insight into the considerations involved in pursing academic medicine, as well as the benefits of formal research training to medical practice. Each of these trainees pursued further academic training at different times in their careers, drawn to academia by a passion for research, critical thinking, social justice and the influence this would have on their career path (Table 1). Similarly, many trainees also felt that time duration and financial constraints were drawbacks of pursuing research. This is extremely relevant now, given the recent funding changes in the Canadian Institute of Health Research for MD/PhD programs. Namely, as of 2016, the CIHR has discontinued funding of their thirty-year old MD/PhD studentship program, in universities across Canada21. Though an MD/PhD is not for everyone, it is unfortunate that this program has been cancelled, likely resulting in a decrease in crossover between medical practice and research. Undoubtedly, the MD/PhD programs in Canada cannot continue as they were before.\n\nFunding matters aside, there is an interesting gender difference in terms of academic medicine engagement. Multiple studies and focus groups have tried to characterize the values and reasons behind the relative lack of female trainees and mentors in academic medicine. This warrants further attention as females likely share the same personal values as their male colleagues, that will draw them to academic medicine, yet additional deterrents have been identified within this group.\n\n\nConclusion\n\nThe personal values that draw one to academic medicine can be used to improve recruitment to academic programs. More research is needed on definition and classification of these personal values, but “intellectual stimulation” is the most consistently identified. Trainees that have a passion for research and academic advancement can be encouraged along this path by identifying them and pairing them with a strong mentor early in their careers. All trainees emphasized the value of mentorship in academia, and its foundation in making or breaking a career in research. Alternatively, we should also be aware of the very real deterrents that are turning away qualified trainees. Long training periods, a lack of autonomy, and financial considerations were identified as deterrents from a career in academic medicine. Being aware of these perceived barriers allows policy makers to address them and help to recruit the best trainees through modification of existing programs.\n\nAt a time when the public’s knowledge of basic science and fundamental medicine is lagging relative to the rapid development of medical science, technology, and social policy22, those who pursue academic medicine will be the essential communicators who bridge the gap. It is paramount that these individuals and groups are identified, supported and lauded for their intellectual thirst. Understanding the personal values and constellation of factors which help an individual decide on a career in academic medicine will hopefully streamline access to academic positions which suit the trainee. This in turn will likely produce medical, scientific, and health policy advancements which will efficiently shorten the “knowledge translation” gap. Overall, this bodes well for patient care at the individual level and for society at large.", "appendix": "Author contributions\n\n\n\nDr. Marissa Tsoi was the primary author of this work, and conducted the literature search and interviews. She guided the overall vision of this article and was responsible for writing it at all stages. Dr. Braden Teitge and Dr. Chris Madan conceived the idea of the paper and were responsible for all subsequent edits. Dr. Teitge also helped to coordinate the process of collaboration and formatting. Dr. Louis Francescutti provided guidance on the article’s overall concept.\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 would like to acknowledge the contributions of all the interviewees, and Dr. Matthew Rose from the University of Alberta for his contribution, guidance, and feedback on this article.\n\n\nReferences\n\nAcademic Medicine. Royal College of Physicians. London, United Kingdom; [Accessed January 2, 2016]. Reference Source\n\nStraus SE, Straus C, Tzanetos K, et al.: Career choice in academic medicine: systematic review. J Gen Intern Med. 2006; 21(12): 1222–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHistory of the MD/PhD program. University of Toronto. Toronto, Ontario; 2016. Reference Source\n\nAdmission requirements of Canadian Faculties of Medicine: admission in 2014. The Association of Faculties of Medicine of Canada. Ottawa, Ontario; [Accessed January 2, 2016]. Reference Source\n\nJagsi R, Guancial EA, Worobey CC, et al.: The “gender gap” in authorship of academic medical literature--a 35-year perspective. N Engl J Med. 2006; 355(3): 281–7. PubMed Abstract \| Publisher Full Text\n\nBorges NJ, Navarro AM, Grover A, et al.: How, when, and why do physicians choose careers in academic medicine? A literature review. Acad Med. 2010; 85(4): 680–6. PubMed Abstract \| Publisher Full Text\n\nBallios BG, Rosenblum ND: Challenges facing physician scientist trainees: a survey of trainees in Canada’s largest undergraduate and postgraduate programs in a single centre. Clin Invest Med. 2014; 37(5): E268–83. PubMed Abstract\n\nFunding Opportunity Details (MD/PhD Students, 2014–2015). ResearchNet. Public Works and Government Services Canada. Ottawa, ON; 2016. [Accessed January 2, 2016]. Reference Source\n\nDesjardins L, King D: Expectations and labour market outcomes of doctoral graduates from Canadian universities.Culture, Tourism and the Centre for Education Statistics. Statistics Canada, Human Resources and Skills Development Canada. Ottawa, Ontario; 2016. [Accessed January 2, 2016]. Reference Source\n\n2013–2016 PARO-CAHO agreement. Professional Association of Residents of Ontario. Toronto, Ontario; 2016. [Accessed January 2, 2016]. Reference Source\n\nJagsi R, Griffith KA, Stewart A, et al.: Gender differences in salary in a recent cohort of early-career physician-researchers. Acad Med. 2013; 88(11): 1689–99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeonard JC, Ellsbury KE: Gender and interest in academic careers among first- and third-year residents. Acad Med. 1996; 71(5): 502–4. PubMed Abstract\n\nReiser LW, Sledge WH, Fenton W, et al.: Beginning careers in academic psychiatry for women--“Bermuda Triangle”? Am J Psychiatry. 1993; 150(9): 1392–7. PubMed Abstract \| Publisher Full Text\n\nBrown AJ, Swinyard W, Ogle J: Women in academic medicine: a report of focus groups and questionnaires, with conjoint analysis. J Womens Health (Larchmt). 2003; 12(10): 999–1008. PubMed Abstract \| Publisher Full Text\n\nFang D, Meyer RE: Effect of two Howard Hughes Medical Institute research training programs for medical students on the likelihood of pursuing research careers. Acad Med. 2003; 78(12): 1271–80. PubMed Abstract \| Publisher Full Text\n\nAndriole DA, Jeffe DB, Hageman HL, et al.: Variables associated with full-time faculty appointment among contemporary U.S. Medical school graduates: implications for academic medicine workforce diversity. Acad Med. 2010; 85(7): 1250–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeng L, Ruzai-Shaprio C: Factors that influence radiologists’ career choices. Acad Radiol. 2003; 10(1): 45–51. PubMed Abstract \| Publisher Full Text\n\nNeacy K, Stern SA, Kim HM, et al.: Resident perception of academic skills training and impact on academic career choice. Acad Emerg Med. 2000; 9(12): 1408–15. PubMed Abstract \| Publisher Full Text\n\nWyrzykowski AD, Han E, Pettitt BJ, et al.: A profile of female academic surgeons: training, credentials, and academic success. Am Surg. 2006; 72(12): 1153–9; discussion 1158–9. PubMed Abstract\n\nShea JA, Stern DT, Klotman PE, et al.: Career development of physician scientists: a survey of leaders in academic medicine. Am J Med. 2011; 124(8): 779–87. PubMed Abstract \| Publisher Full Text\n\nSilverman M: Cutting funds for MD/PhD programs a blow to Canadian innovation. The Globe and Mail, 2015; Accessed February 1, 2016. Reference Source\n\nMorris ZS, Wooding S, Grant J: The answer is 17 years, what is the question: understanding time lags in translational research. J R Soc Med. 2011; 104(12): 510–520. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "20201", "date": "24 Feb 2017", "name": "Kirstie J. Whitaker", "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\nSynopsis Tsoi and colleagues start with a definition of academic medicine and a summary of this career path and who chooses to take it. They then explain the pros and cons of academic medicine as have been described in the currently published literature. Finally, they present summaries of interviews conducted with five medical professionals: two MD students, one PhD student, one MD/PhD student and one experienced clinician (MD) who has returned to complete a PhD at an advanced stage of his career.\n\nCompliments It is important for personal motivations and values to be included in our understanding of career paths in all aspects of science and medicine. This article has the potential to take “common knowledge” and hearsay and put it into a peer-reviewed literature. It makes these discussions findable and citable: important steps towards progressing our understanding of addressing structural inequalities in career progression. This work stands to benefit many readers who are interested in careers in academic medical but are looking for more detailed information on the prospect.\n\nConcerns/Suggestions Audience Who is the intended audience for this opinion piece? The abstract mentions trainees, educators and policy makers, and the authors are right that this topic is of interest to all these groups. Unfortunately, the focus for each of them are quite different and I think the paper fails to provide succinct take home messages for any of them. (Alternatively, this is a qualitative research article and should be structured as such, see my comments below). I’d recommend developing the discussion section and potentially including actions that interested parties may take as a result of the findings presented in the paper. To be clear: my recommendation is to restructure the opinion article, as there is a lot of useful advice/information included in the introduction that I think could be put to better use supporting the research findings, rather than add in more information to the discussion.\n\nIntegrating personal values The introduction to this opinion article outlines most of the arguments for/against a career in academic medicine so the summaries of the interviews do not end up providing much new information to the reader. If the point of the article is to present personal values, I’d recommend integrating quotes from the interview subjects along with the background literature. Although not necessary, I suspect that arranging the information around themes rather than by individual participant would synthesise the findings more effectively. Qualitative research is not my area of expertise but I enjoyed these suggestions from London School of Economics (which includes links to further reading on the topic).\n\nMissing voices There are key demographics who are not interviewed in this article on the career path in academic medicine: specifically, those of clinicians who do not see the benefits of it and/or who those who were not able to continue along this path. Furthermore, all people interviewed are students which leaves out most of the time spent along this career path. This is fine, but I’d like the authors to acknowledge that they are presenting a biased sample which may limit its generalisability for readers considering a career in academic medicine. Related to this point, and more minor in my opinion, is that this article seems to be written about academic medicine careers in Canada. I’d recommend that the authors clarify this focus in the abstract and either acknowledge that these findings may not be relevant to clinicians in other countries or develop the breadth of the introduction and discussion to consider these differences.\n\nFemales Please do not refer to adult women as “females”. The use of this adjective as a noun reduces women to their reproductive abilities and diminishes their humanity (see Jezebel for my favourite article on the subject). I’m confident that this was not the authors’ intention, but given that a focus of this opinion article is the reasons women are not well represented within academic medicine, I’d advise avoiding further alienation. I’d recommend referring to women and men (rather than males) as appropriate.", "responses": [] }, { "id": "20199", "date": "27 Feb 2017", "name": "Tobias C. Wood", "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 opinion article consists of two parts.\nThe first is a thorough literature review. It gives a solid overview of the issues surrounding the MD/PhD program. I have no issues with this section.\nThe second part consists of five transcribed interviews, and I consider it weaker than the first section. The evidence presented is only anecdotal, with a very small sample size. The author states that the interviewees were drawn from the author's academic contacts, so although a range of views and backgrounds is represented, sample bias cannot be excluded. Given that this an opinion and not a full research article this is not a reason for rejection, but this section could be greatly strengthened with additional interviews, ideally from outside the author's network. Alternatively, given the length of time that has elapsed since the original interviews, follow-up interviews checking whether the participants still felt the same way about their courses would be enlightening.\nThe discussion and conclusion sections are mostly good. However, the second paragraph of the discussion contains the following sentence: \"Multiple studies and focus groups have tried to characterize the values and reasons behind the relative lack of female trainees and mentors in academic medicine.\", but no citations are given. I suspect that the authors are correct in this assertion, but a relevant citation or other evidence to back up this statement must be added before indexing.", "responses": [] } ]	1	https://f1000research.com/articles/5-1903
https://f1000research.com/articles/7-487/v1	24 Apr 18	{ "type": "Research Article", "title": "Individual typological differences in a neurally distributed semantic processing system: Revisiting the Science article by Mitchell et al. on computational neurolinguistics", "authors": [ "Hiroyuki Akama" ], "abstract": "Background: Revisiting the 2008 Science article by Mitchell et al. on computational neurolinguistics, individual typological differences were found as striking characteristics in the patterns of informative voxels crucial for the distributed semantic processing system. Methods: The results of different feature selection methods (ANOVA and Stability) were compared based on the open datasets of each subject for evaluating how these features were decisive in predicting human brain activity associated with language meaning. Results: In general, the two selection results were similar and the voxel-wise ranks were correlated but they became extremely dispersive for a subgroup of subjects exhibiting mediocre precision when examined without regularization. Quite interestingly, looking at the anatomical location of these voxels, it appears that the modality-specific areas were likely to be monitored by the Stability score (indexing “identity”), and that the ANOVA (emphasizing “difference”) tended to detect supramodal semantic areas. Conclusions: This minor finding indicates that in some cases, seemingly poor data may deeply and systematically conceal information that is significant and worthwhile. It may have potential for shedding new light on in the controversy pertaining to cognitive semantics, which is divided into modality-biased (embodied) and amodal symbol theories.", "keywords": [ "fMRI", "MVPA", "feature selection", "individual variability", "semantics" ], "content": "Introduction\n\nIt is widely acknowledged that despite some challenges in multi-voxel pattern analysis (MVPA), the issue of individual variability, raised as a penalty to classification accuracy in cross-subject modelling, is challenging to overcome, particularly when targeting concepts and meanings conveyed by language1. Admittedly, the precision rates in MVPA could be mostly uniform for experiments successfully performed at the individual first level, as in the case of the classically recognized Science article authored by T. Mitchell and his group (Predicting Human Brain Activity Associated with the Meanings of Nouns)2. In their study, distributed brain activation patterns were observed in nine subjects conceptualizing 60 concrete nouns in an fMRI scanner, and these neural patterns were regressed on a co-occurrence probability in a text corpus between each of these fMRI nouns and semantic features (25 basic verbs). The highly significant prediction accuracy that they obtained at the subject level was associated with a pattern separability built on a set of informative voxels as features, which are distributed across numerous brain areas and differ across subjects. The Science study and other computational neurolinguistic reports3–7 could generate classifiers with high precision (owing to the L2-regularization technique that obliterates individual variability) and draw plausible semantic maps in the individual brain.\n\nHowever, functionality at work in the individual brain remains to be specified further beyond unanimously acceptable modelling results. In this study, an alternative view is put forward to uncover systematicity and provide typology in individual variability through a reanalysis of the open data used in the Mitchell et al.’s Science study. Several previous studies have reported that despite the almost invariably accurate performance of the subjects in conceptualization tasks, a fundamental difference is seen in the magnitude of how the information on the feature voxels is dispersive based on their anatomical locations1,5. Unquestionably, the experimental paradigm developed by Mitchell et al. hinged on modality specific factors, which promoted distributiveness of semantic processing systems. Their stimulus set, which used captioned drawings with considerable visual effects and concrete nouns with implications of some motion/perception, rendered the experiment sensitive to embodied cognition8. The attribute generation task, which consisted of thinking about the properties of the object, could allow free association and perceptual simulation9. Yet, even without accounting for the modality specific factors, there remains some debate pertaining to the supramodal semantic centers in general as to whether the left temporal pole and anterior temporal gyrus (hub and spoke model)10–11 or the left middle temporal gyrus and the left angular gyrus (high level convergence model)12–13 are nodal for the loci of a genuinely semantic process. The primary goal of the present study was to determine whether such topology of semantic processing can be elucidated through a typology of fMRI subjects and to examine how that typology is determined by the subjects’ hidden neural responses characteristic to the selection of the most informative voxels.\n\n\nMethods\n\nThe datasets were nine .mat files corresponding to the nine subjects (P1–P9), downloaded from the website of Carnegie Mellon University. A ‘.mat’ file was created (by using MATLAB R2015a) from each subject’s data, called 'runByVoxByNouns-P<number>.mat,' as a three-dimensional array corresponding to x: runs (six repeated presentations) by y: voxels by z: words (60 fMRI nouns). This procedure facilitated the computation of informative voxel locations identified by different feature-selection methods, which were the F values of ANOVA to measure and evaluate the mean value across the items repeatedly presented in a learning set and the Stability scores to distinguish voxels that exhibited consistently similar activation patterns to the items for machine learning14. Mitchell et al. adopted the latter method to select the top 500 voxels, which involves computing the Pearson's correlation coefficient of the activation vectors for the stimulus nouns over 15 (=6P2) pairs of fMRI presentation runs repeated 6 times. They computed the average pairwise correlation for each voxel over all pairs of rows in the matrix composed of six presentation runs by 58 nouns (reserving two nouns for testing). In this study, cross-validation was not performed except for computing the modeling accuracy for each subject using the ordinary least square method (OLS) without L2-regularization. The top 500 voxels were selected from the overall runs by the ANOVA and the Stability scores, and the number of the voxels not shared by the two selection results (subtraction operation for the two sets, i.e., type 1: ANOVA set – Stability set and type 2: Stability set – ANOVA set) was counted as “divergence” for each subject, as shown in Table 1. The voxel-wise ranks in the two selection results were compared with each other with Spearman’s rank correlation coefficient and the subject-wise rho and the corresponding p values were computed. The ANOVA and Stability feature-ranking data were all mapped to anatomical regions according to the automated anatomical labeling (AAL) atlas15.\n\nThe top 500 voxels were selected by the ANOVA and the Stability and the modelling accuracy based on the ordinary least square method (OLS) without adjustment of L2-regularization. The subject-wise ‘rho’ and the corresponding p values represent Spearman’s rank correlation coefficient between the voxel-wise ranks in the two feature selection results. ‘Divergence’ implies the number of the selected voxels extracted by the subtraction operation of ANOVA set – Stability set or Stability set – ANOVA set.\n\n\nResults\n\nThe raw modelling accuracy, based on the ordinary least square methods (OLS) without L2-regularization, decayed with the magnitude of \"divergence\" between the ANOVA and the Stability score and with the decrease in rank similarity between the voxels selected by each method (Table 1). Admittedly, good modelling accuracy is associated with high F values and Stability scores for the selected voxels, as is seen in P1 eliciting the best precision by a wide margin and the next best group of P2–P4. However, there was no mean difference in feature scores among the subjects of the middle group, P5, P6, and P7, although P5 and P7 exhibited significant Spearman’s rank correlation coefficients, divergence less than 100, and OLS precision higher than 70%, while none of these conditions was true for P6. When visualizing the score distributions of the top 500 voxels with a notched box plot, it is apparent that the poor-performance group (P6, P8, and P9) was characterized by narrowness of the boxes and the low upper whiskers (Figure 1) and dispersiveness of the voxel-wise ranks in the two selection results (Figure 2).\n\nThe voxels were selected by the ANOVA (above) and the Stability score (below). The horizontal axis corresponds to the numbered participants. This figure was depicted using the MATLAB function of ‘boxplot.’ Data points exceeding the whiskers are displayed using +. The singularity of Participant 1 is conspicuous with the best scores by far, while the typology of the other participants was easily recognized classifying them into two groups (fair: P2, P3, and P4; poor: P5–P9). However, a finer-grained observation allows us to stress the narrowness of the boxes and the low upper whiskers for P6, P7, and P9 and bundle them as a small subgroup of interest.\n\nThe residuals to the regression line of y=x represent how closer or farther the two kinds of ranks of the selected voxels. The dots right to the red vertical line (y = 500) represent the voxels (type 1) selected by the ANOVA but not by the Stability score as the top 500. The dots over the red horizontal line (x = 500) correspond to the voxels (type 2) selected by the Stability score but not by the ANOVA under the same criteria. Irregularity in distribution is found in P6, P8, and P9.\n\n\nDiscussion\n\nOur results support the notion that particular types of individuals differ markedly in their way of recruiting voxels with respect to different feature selection methods, i.e., Stability scores and F values of ANOVA. The Stability scores examine the extent to which each voxel reacted to the same stimulus across runs in a constant manner; therefore, it is the “identity” of an object that is emphasized by this index as invariable through repetition. Conversely, the F values of ANOVA pertain to the magnitude of between-group variance across the responses to the 60 nouns with respect to the within-group variance across the 6 presentations of each individual noun; therefore, the “difference” is likely to be captured by that index although it should be inextricably linked with the “identity” side. In consequence, regardless of the feature selection method, mostly the same voxels with a similar top 500 ranking order could be selected from the brains of P1–P5 and P7, but the remaining subjects (P6, P8, and P9) showed important divergence from the list of the 500 important voxels selected by the two methods. The mean index values for the top voxels were significantly larger in the former subjects than the latter ones for each method; the difference in raw classification accuracy without the regularization effect was conspicuous between these subject groups. However, an in-depth analysis revealed some questions to be delineated, since P5 and P7 may be treated equally as members of an interesting subgroup in that, despite the highly significant rank correlation for the selected voxels, the mean values were relatively low and not significantly different from those of P6 in the poor-performance group.\n\nWhen tapping into the anatomical regions from which feature voxels were selected, the most intriguing property was that high precision in modelling was guaranteed rather by extra-linguistic regions. It is noteworthy that in P1 (recording 82% as classification accuracy by the OLS with no regularization), the majority of the top 487 informative voxels shared by the Stability score and the ANOVA were found in the visual areas of the temporal and occipital lobes and several in the frontal and parietal lobes. Differently from all the other subjects, the left inferior frontal gyrus, pars triangularis (\"Frontal_Inf_Tri_L\"), and the left precuneus (\"Precuneus_L\"), frequently considered as involved in executive functions of language activity, were not recorded in the overlapping selected voxel areas of P1. When focusing on the poor-performance subject group (P6, P8, and P9), which exhibited a large divergence (larger than 1 standard deviation from the mean) between the voxel selections by the two methods, it appeared that the modality-specific areas were likely to be monitored by the Stability score (indexing “identity”), and that the ANOVA (emphasizing “difference”) tended to detect supramodal semantic areas. The voxels type 1 (selected by the ANOVA but not by the Stability score as the top 500) and type 2 (selected by the Stability score but not by the ANOVA under the same criteria) voxels were mostly extracted from different anatomical regions. The frequency distribution tables (Figure 3) represent the number of type 1 and type 2 voxels selected from P6, P8, and P9 and attributed to each anatomical area in the AAL brain atlas. The ANOVA highlighted as locations of type 1 voxels, some areas for amodal or supramodal semantic processing, especially the left middle temporal gyrus (“Temporal_Mid_L”) which was the most populous by far in this category. In contrast, the Stability score tended to introduce bias to the vision-related areas, notably the left middle occipital gyrus (“Occipital_Mid_L”), which may reflect stimulus modality or perceptual symbols in embodied cognition.\n\nThe voxels were classified into type 1 (above, ANOVA subtracted from the Stability score) and type 2 (below, Stability score subtracted from the ANOVA) and attributed to each anatomical area in the AAL brain atlas (the voxels labelled 'Not_Found' were removed from the lists). The type 1 voxels tend to belong to the supramodal semantic regions (such as “Temporal_Mid_L”), whereas the type 2 ones are characterized by the dominance of the visual area (such as “Occipital_Mid_L”).\n\nThis divergence allows us to shed a new light on a traditionally controversial subject in neural semantics; where is the border that separates the brain regions selective to purely conceptual functions and sensory-driven, modality-dominant, so extrinsic to meaning processing? The compatibility of the Stability score with the perceptual modalities may suggest with the embodiment view (to which Mitchell et al. also referred for the neural signature of the verb “eat”) that the “identification” of a concept is materially founded upon a sensory-perceptual system and real-life experience with its instances of referent (or stimuli) to shape its cognitively grounded symbols. However, the voxel information brought by the ANOVA enables us to propose an alternative view for the discrimination power in language, having an affinity for the amodal (not to say disembodied) symbol theory. Descended from the school of Saussure, this theory (often relying on lexical co-occurrence information from language corpora as in the case of the Science study) postulates that the value of a symbol (or a linguistic sign) is not derived from its intrinsic sense but from language itself as a computability system of “difference.” It is quite intriguing that the reanalysis of the Science data assessed, through the variability of subjects performing a language task, a salient discrepancy between the brain regions informative of manifold essence in semantic processing. Indeed, we are not yet in a position to argue these philosophically opposite views only through a succinct review such as this report. However, at least we may conclude here that such a fundamental issue was, quite interestingly, readdressed by reanalyzing the data from a subject group that elicited inconsistency to the feature selection methods and relatively low precision rates to fMRI machine learning classifiers.\n\n\nData availability\n\nThe dataset of Mitchell et al. was downloaded from\n\nhttp://www.cs.cmu.edu/afs/cs/project/theo-73/www/science2008/data.html\n\nDataset 1: Reanalysis of Mitchell et al. data. 10.5256/f1000research.14584.d20176716", "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\nAcknowledgement\n\nThe author would like to thank Editage (www.editage.jp) for English language editing.\n\n\nReferences\n\nAkama H, Murphy B: Emerging Methods for Conceptual Modelling in Neuroimaging. Behaviormetrika. Springer. 2016; 44(1): 117–133. Publisher Full Text\n\nMitchell TM, Shinkareva SV, Carlson A, et al.: Predicting human brain activity associated with the meanings of nouns. Science. 2008; 320(5880): 1191–5. PubMed Abstract \| Publisher Full Text\n\nBullinaria JA, Levy JP: Limiting factors for mapping corpus-based semantic representations to brain activity. PLoS One. 2013; 8(3): e57191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnderson AJ, Binder JR, Fernandino L, et al.: Predicting Neural Activity Patterns Associated with Sentences Using a Neurobiologically Motivated Model of Semantic Representation. Cereb Cortex. 2017; 27(9): 4379–4395. PubMed Abstract \| Publisher Full Text\n\nPereira F, Detre G, Botvinick M: Generating text from functional brain images. Front Hum Neurosci. 2011; 5: 72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkama H, Miyake M, Jung J, et al.: Using Graph Components Derived from an Associative Concept Dictionary to Predict fMRI Neural Activation Patterns that Represent the Meaning of Nouns. PLoS One. 2015; 10(4): e0125725. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuth AG, de Heer WA, Griffiths TL, et al.: Natural speech reveals the semantic maps that tile human cerebral cortex. Nature. 2017; 532(7600): 453–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWillems RM, Casasanto D: Flexibility in embodied language understanding. Front Psychol. 2011; 2: 116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEngelen JA, Bouwmeester S, de Bruin AB, et al.: Perceptual simulation in developing language comprehension. J Exp Child Psychol. 2011; 110(4): 659–75. PubMed Abstract \| Publisher Full Text\n\nPatterson K, Nestor PJ, Rogers TT: Where do you know what you know? The representation of semantic knowledge in the human brain. Nat Rev Neurosci. 2007; 8(12): 976–87. PubMed Abstract \| Publisher Full Text\n\nRice GE, Lambon Ralph MA, Hoffman P: The Roles of Left Versus Right Anterior Temporal Lobes in Conceptual Knowledge: An ALE Meta-analysis of 97 Functional Neuroimaging Studies. Cereb Cortex. 2015; 25(11): 4374–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBinder JR, Desai RH, Graves WW, et al.: Where is the semantic system? A critical review and meta-analysis of 120 functional neuroimaging studies. Cereb Cortex. 2009; 19(12): 2767–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBinder JR, Desai RH: The neurobiology of semantic memory. Trends Cogn Sci. 2011; 15(11): 527–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPereira F, Mitchell T, Botvinick M: Machine learning classifiers and fMRI: a tutorial overview. NeuroImage. 2009; 45(1 Suppl): S199–209. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTzourio-Mazoyer N, Landeau B, Papathanassiou D, et al.: Automated anatomical labeling of activations in SPM using a macroscopic anatomical parcellation of the MNI MRI single-subject brain. NeuroImage. 2002; 15(1): 273–89. PubMed Abstract \| Publisher Full Text\n\nAkama H: Dataset 1 in: Individual typological differences in a neurally distributed semantic processing system: Revisiting the Science article by Mitchell et al. on computational neurolinguistics. F1000Research. 2018. Data Source" }	[ { "id": "33424", "date": "18 Jul 2018", "name": "Chiu-Hsieh Hsu", "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 manuscript is a reanalysis of the study of Mitchell et al. (2008). The author claims that using different feature selection methods can help to identify areas that are functional distinctive in individual level. The finding of the presented study provides substantial implication to studies that focus on how neural activation encoding the semantic content. I only have few minor comments.\n(on page 3) please provide details of the cross-validation method of the OLS analysis.\n\nIn Discussion, the author describe about the distribution of \"type 1 voxels\" and \"type 2 voxels\" in some participants. I wonder if the results in the rest of the participants show a similar pattern?\n\nI would like to suggest that the author can provide figures or tables that show the distribution of three types of voxels (type 1, type 2, and convergent voxels) of each participant. This information would help the reader to see the consistency/divergence of encoding models in each participant.\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/7-487
https://f1000research.com/articles/7-485/v1	23 Apr 18	{ "type": "Opinion Article", "title": "VALIDATE: Exploiting the synergy between complex intracellular pathogens to expedite vaccine research and development for tuberculosis, leishmaniasis, melioidosis and leprosy", "authors": [ "Helen A. Fletcher", "Mitali Chatterjee", "Andrea Cooper", "Tracy Hussell", "Paul M. Kaye", "Joann Prior", "Rajko Reljic", "Samantha Vermaak", "Martin Vordermeier", "Ann Williams", "Helen McShane", "Mitali Chatterjee", "Andrea Cooper", "Tracy Hussell", "Paul M. Kaye", "Joann Prior", "Rajko Reljic", "Samantha Vermaak", "Martin Vordermeier", "Ann Williams", "Helen McShane" ], "abstract": "For several complex intracellular pathogens, we have an urgent need for effective vaccines and yet there are common barriers to vaccine development. These diseases, including tuberculosis, leishmaniasis, leprosy and melioidosis, cause a huge burden of disease and disproportionately affect low and middle income countries. They are therefore often neglected due to the marginalisation of affected populations and the poor predicted commercial return on investment. Barriers to vaccine development include an incomplete understanding of protective immunity and translation from the bench into clinical vaccine trials. The current linear approach to vaccine research and development for these pathogens, which involves basic research, vaccine design, and vaccine evaluation in preclinical challenge models and clinical trials, is inefficient for these complex intracellular pathogens. We have established a Global Challenges Research Fund Network for VAccine deveLopment for complex Intracellular neglecteD pAThogEns, “VALIDATE”, where we aim to adopt a more flexible, integrated cross-pathogen approach to accelerate vaccine research and clinical development for these four pathogens, by cross-pathogen analyses, cross-discipline collaborations, and repeated integration of data from human and animal studies. This network provides a unique opportunity to bring together individuals working on four exemplar complex intracellular neglected pathogens (M.tb, Leishmania spp., B. pseudomallei and M.leprae), which share a common lifestyle as pathogens of macrophages, induce similar end-stage pathologies and alter host immune and metabolic responses. The horizontal collaborations established throughout this network, together with the provision of a protected environment for early data sharing, will exploit these biological synergies. By interrogating mechanisms that lead from infection to disease, we will be able to develop common vaccine development strategies for these and other complex intracellular pathogens.", "keywords": [ "Tuberculosis", "TB", "vaccine", "leishmaniasis", "leprosy", "melioidosis", "neglected", "intracellular" ], "content": "Introduction\n\nThe global burden of disease and death caused by Mycobacterium tuberculosis (M.tb), Leishmania spp, Burkholderia pseudomallei and M.leprae is enormous. Tuberculosis (TB) kills more people than any other infectious disease, with 1.7m deaths and 10.4m new cases in 20161. The significant economic impact of TB in low- and middle income countries (LMICs) is due to the disproportionate involvement of the most economically active young adults. Furthermore, bovine TB has a very significant effect on health and economic development in LMIC2,3. The leishmaniases represent a group of heterogeneous diseases caused by intracellular protozoan parasites of the genus Leishmania. They are recognized by the WHO as major neglected diseases of poverty, and disproportionately affect populations in LMICs. Approximately 1.5m new cases occur each year, across 98 countries worldwide, with 20,000–40,000 deaths4. Canine visceral leishmaniasis is not only a veterinary problem but a significant reservoir for human disease, notably in Brazil and in countries bordering the Mediterranean. As with TB, leishmaniasis and poverty exist in a vicious circle, with the economic impact for patients, families and communities well documented.\n\nLeprosy and melioidosis also compromise economic productivity in LMICs, are difficult to treat and are in need of effective vaccination strategies. In 2016, there were 216,108 new leprosy cases registered globally5. 14 countries contain 95% of these globally reported cases, all of which are in LMICs. Of these, India has the greatest number of cases (59%), followed by Brazil (14%) and Indonesia (8%). Although the number of cases worldwide continues to fall, pockets of high prevalence remain in certain areas such as Brazil, South Asia (India, Nepal, Bhutan), some parts of Africa (Tanzania, Madagascar, Mozambique), and the western Pacific. Melioidosis is a disease caused by the Gram-negative soil-dwelling bacterium Burkholderia pseudomallei6,7. The estimated global burden of melioidosis is 165,000 human melioidosis cases/pa, causing 89,000 deaths7. The burden of disease is in South East Asia, where the in-hospital mortality is ~40%8. These estimates of incidence for leprosy and melioidosis are likely to be significant under-estimates. The development of effective vaccines against any or all of these pathogens would have a significant health benefit around the world.\n\n\nWhat does VALIDATE aim to achieve?\n\nThe lack of rapid, coordinated dissemination of information between research groups hinders vaccine development. Our strategy is to establish a network of multi-disciplinary scientists across the UK and LMICs, who work on vaccine research and development for these four complex intracellular neglected pathogens. We will take an interdisciplinary approach with immunologists, clinicians, social scientists, veterinarians, epidemiologists, bioinformaticians, mathematical modellers, and animal model experts to overcome barriers to progression of vaccine development. The coordinated, integrated and iterative sharing of data to define protective immunity in animal models, in target animal species, and in human experimental medicine studies, across complex intracellular pathogens will expedite the development of effective vaccines. Our particular focus is on building and strengthening cross-pathogen, cross-species, cross-discipline and cross-continent collaborations, to foster novel insights and new perspectives that lead to an increased understanding of the nature of protective immunity. Currently, few horizontal collaborations occur between these distinct pathogen research fields. VALIDATE will add value by establishing horizontal collaborations whereby innovative research solutions in one field will be rapidly disseminated to the other fields. This interaction will leverage progress in one field and promote accelerated identification of immune mechanisms of protection and evaluation of vaccine candidates in human and animal models. Given the many common immunological and microbiological features of the complex intracellular pathogens selected as the focus of VALIDATE, these newly-formed horizontal cross-pathogen collaborations will yield novel insights that will be explored iteratively in in vitro and in vivo experiments in animal models and human experimental medicine studies. For example, a key feature of both TB and leishmaniasis is granuloma formation. Imaging and transcriptomics have been used to define the Leishmania granuloma; these techniques could be further used to interpret the TB granuloma9,10.\n\nTo date, VALIDATE has 114 members from 24 countries, 17 of these LMICs, and this range of knowledge and experience will broaden our scope and ensure relevance. The VALIDATE members are selected for their complementary scientific expertise and range from internationally recognised leaders in their field to Early Career Researchers (ECRs), with students and members of the general public who are interested in vaccine research also welcome. All scientific members have significant track records in research into TB, leishmaniasis, leprosy or melioidosis. Members can come from academia, governmental organisations, non-profits, and industry. Specific fields of interest include basic immune mechanisms in humans and preclincial animal models, mucosal immunity, immune correlates of protection, immunopathogenesis, translational vaccine development, mathematical modelling and social science aspects of vaccinology.\n\n\nHow will we do it?\n\nVALIDATE has four main activity streams.\n\nFirstly, we are providing relevant funding to our members: as pump-priming grants of £20–50k for innovative collaborative research projects; training grants to enable our ECRs to attend courses, workshops and laboratory exchanges useful for their career progression; and as ECR Fellowships to springboard these Fellows to scientific independence as new group leaders.\n\nSecondly, VALIDATE is developing a dedicated data-sharing portal for members only, which will encourage real-time sharing of data, catalysing the application of insights from one field into another. The VALIDATE Research Data Analyst is tasked with actively searching for data synergies and differences where researchers working on different pathogens can learn from another, and works with members on existing and arising datasets.\n\nThirdly we provide Continuing Professional Development (CPD) opportunities for our members, including workshops on areas of mutual interest, seminars (that are live-streamed online so that overseas members can also benefit), and a mentoring scheme for ECRs and early PIs open to members across the world – we have linked five mentees with their chosen mentors in our first round. Scientific research is a valuable economic activity to the host country, and we will build sustainable human resource capacity within both the UK and low- and middle-income member countries.\n\nFinally, we are developing a vibrant and interactive network, facilitating the formation of new collaborations and ideas, and speeding the dissemination of useful information amongst our members using the full range of communications tools. We have created a hub website (www.validate-network.org) where our members can easily find information about new research and papers, relevant funding calls, events, and training, mentoring and other opportunities of interest. Interested parties can read about our funded work to date, while a searchable directory of members on our website is facilitating the formation of new collaborations. Our social media (@NetworkValidate) raises awareness of these four pathogens and VALIDATE’s research through engagement of members, other scientists and the lay public. An annual meeting, free to all members, furthers our outreach, and boosts existing and potential collaborations. There are travel scholarships available for up to seven LMIC members each year to facilitate the broadest level of attendance.\n\n\nSummary\n\nVALIDATE provides a unique opportunity for an interdisciplinary and integrated approach to vaccine research and development for these four exemplar complex intracellular neglected pathogens. Sustainable collaborations are being formed and strengthened by the resources provided, innovative research has been funded, and ECR progression is underway. We are using this platform to accelerate vaccine development for our focus pathogens, and are building our funding portfolio to ensure sustained progress in these critical areas.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Competing interests\n\n\n\nHelen McShane and Helen Fletcher are co-investigators on the VALIDATE grant a GCRF/MRC funded Vaccine Network Grant\n\n\nGrant information\n\nVALIDATE is supported by the Global Challenges Research Fund (GCRF) Networks in Vaccines Research and Development which was co-funded by the MRC and BBSRC. The GCRF is a 5-year £1.5Bn fund established by the UK government.\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\nWorld Health Organization: Global Tuberculosis Report 2016. 2017; World Health Organization: Geneva, Switzerland. Reference Source\n\nAyele WY, Neill SD, Zinsstag J, et al.: Bovine tuberculosis: an old disease but a new threat to Africa. Int J Tuberc Lung Dis. 2004; 8(8): 924–37. PubMed Abstract\n\nAmeni G, Aseffa A, Engers H, et al.: Cattle husbandry in Ethiopia is a predominant factor affecting the pathology of bovine tuberculosis and gamma interferon responses to mycobacterial antigens. Clin Vaccine Immunol. 2006; 13(9): 1030–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlvar J, Vélez ID, Bern C, et al.: Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 2012; 7(5): e35671. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Fact sheet: Leprosy. 2018. Reference Source\n\nPeacock SJ, Limmathurotsakul D, Lubell Y, et al.: Melioidosis vaccines: a systematic review and appraisal of the potential to exploit biodefense vaccines for public health purposes. PLoS Negl Trop Dis. 2012; 6(1): e1488. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLimmathurotsakul D, Golding N, Dance DA, et al.: Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nat Microbiol. 2016; 1: 15008. PubMed Abstract \| Publisher Full Text\n\nLimmathurotsakul D, Wongratanacheewin S, Teerawattanasook N, et al.: Increasing incidence of human melioidosis in Northeast Thailand. Am J Trop Med Hyg. 2010; 82(6): 1113–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoore JW, Moyo D, Beattie L, et al.: Functional complexity of the Leishmania granuloma and the potential of in silico modeling. Front Immunol. 2013; 4: 35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeattie L, Sawtell A, Mann J, et al.: Bone marrow-derived and resident liver macrophages display unique transcriptomic signatures but similar biological functions. J Hepatol. 2016; 65(4): 758–768. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "33590", "date": "02 May 2018", "name": "Antonio Campos-Neto", "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 reports the goals of the recently created initiative named VALIDATE, which is an organization funded by the Global Challenges Research Fund (GCRF) Networks in Vaccines Research and Development, which is co-funded by the The Medical Research Council (MRC) and The Biotechnology and Biological Sciences Research Council (BBSRC).\nVALIDATE is a fantastic Network that aims to promote vaccine research and development for complex intracellular pathogens that cause significant disease burden in low and middle-income countries (LMIC). The authors’ summary of VALIDATE activities is outstanding.\nThe authors and founding members of this much needed initiative deserve the compliments of scientific communities and Government authorities of not only of LMICs but also of developed countries.\nOne minor suggestion is to add in the article a sentence or two indicating the plans for long term funding sustainability of VALIDATE.\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": "34293", "date": "29 May 2018", "name": "Lisa A. Morici", "expertise": [ "Reviewer Expertise My area of research focuses on vaccine development against melioidosis." ], "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 comprehensively describes the scope and purpose of VALIDATE, a Global Challenges Research Fund Network. VALIDATE aims to exploit the similarities among complex intracellular bacterial pathogens, including those responsible for tuberculosis, leishmaniasis, melioidosis, and leprosy. Of the target diseases, only tuberculosis receives considerable attention from major funding agencies. Establishment of VALIDATE will help unite researchers from across the globe to accelerate vaccine development against these largely neglected diseases. It will also provide a resource for data sharing, pilot funding, and mentorship for young investigators.\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": "34153", "date": "26 Jun 2018", "name": "Steven G. Reed", "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\nMechanisms of immunity to intracellular pathogens, such as those causing tuberculosis, leishmaniasis, and leprosy have much in common, and as such knowledge in each of these areas should be leveraged and applied as much as possible to these and other intracellular pathogens. The functional definition of CD4 T cell subsets was pioneered in experimental leishmaniasis models and the knowledge has been applied to other infectious diseases, allergy and cancer. Of particular relevance is the selection of vaccine delivery systems and adjuvants that may selectively drive desired immune responses.\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/7-485
https://f1000research.com/articles/7-158/v1	07 Feb 18	{ "type": "Case Report", "title": "Case Report: Cerebral leukodystrophy and the gonadal endocrinopathy: a rare but real association", "authors": [ "Mohammad Humayun", "Abidullah Khan", "Mohammad Humayun" ], "abstract": "A 30 year old married Pakistani woman presented in January 2018 with an eight month history of progressive left sided weakness, ataxia, spasticity, underdeveloped secondary sexual characteristics and primary infertility. She was the elder sister of a 19 year old bed bound woman who was diagnosed with vanishing white matter (VWM) disease 12 months previously. The MRI scan of the brain demonstrated diffuse leukodystrophy and her hormonal assays were significant for premature ovarian failure. Results from her genetic tests demonstrated a point mutation in eukaryotic initiation factor 2B (EIF2B). Thus, she was the second confirmed case of VWM from her family of 12 siblings with normal parents.", "keywords": [ "Vanishing white matter", "neuro-ovarian failure", "leukodystrophy" ], "content": "Introduction\n\nVan der Knaap described a new clinical entity of neuro-ovarian failure in 1996 as vanishing white matter(VWM) disease1. This rare entity is also known as ovarioleukodystrophy and is caused by a mutation of the eukaryotic initiation factor 2B (EIF2B)2. It most commonly presents in infants or early childhood as a progressive central neuronal failure causing limb weakness, spasticity, cognitive decline, seizures, encephalopathy and ultimately death2,3. The onset in adults is very rare and in female patients, is frequently associated with premature ovarian failure3. In March 2017, we reported the first ever case of VWM affecting a 19 year old woman from Pakistan4. Herein, we report the case history of her elder sister diagnosed as VWM in January 2018.\n\n\nCase report\n\nA 30 year old Pakistani woman, presented in January 2018 with an eight month history of progressive left side weakness in pyramidal distribution. She had developed spasticity in the left lower limb and had partial contractures affecting both of her hands. Since the previous month, she felt clumsy in her right arm and leg. She had cerebellar signs and bilateral optic atrophy. However, the rest of her cranial nerves, the spine and the sensory system were normal. She was married for the last four years and had never conceived. She had secondary amenorrhea and underdeveloped secondary sexual characteristics. Her mental state examination was normal. There was no precedent history of any trauma, surgery, malignancy or infection.\n\nHer MRI brain scan was remarkable for diffuse leukodystrophy (Figure 1 and Figure 2). Her hormonal assays were consistent with premature menopause (Table 1). An ultrasound of her abdomen and pelvis was remarkable for small ovaries and uterus. Her lumbar puncture results were normal and there were no oligoclonal bands in her cerebrospinal fluid (CSF). She underwent genetic tests including sequence analysis and polymerase chain reaction (PCR), which demonstrated a point mutation in the EIF2B4 gene. Thus, she was diagnosed as a second case of VWM from the same family of 12 siblings and 2 parents. None of the family members were screened due to affordability issues.\n\nShe was counseled and vaccinated against the common pathogens. She was prescribed baclofen 20mg/day and clonazepam 0.5mg/day for her spasticity. She will be reviewed again in 6 months time. The follow-up plan includes a detailed physical assessment and a repeat MRI brain scan.\n\n\nDiscussion\n\nOvarioleukodysptrophy or vanishing white matter disease (VWM) is a rare autosomal recessive disorder. It has central neuronal presentation in the form of progressive ataxia, spasticity, and variable optic atrophy in combination with endocrinopathy manifesting as premature ovarian failure3,4. This disease has protean spectrums of presentation ranging from the most severe prenatal and infantile forms to the relatively less severe adult onset varieties5. Our second patient had the adult onset disease, a phenotype similar to her younger sister.\n\nThe mildest of all, the adult onset variant of VWM presents with a combination of neurological features including pyramidal weakness, cerebellar signs and optic atrophy in association with premature ovarian failure4,5. The sensory system, the cognitive function and the rest of the cranial nerves are relatively spared, at-least initially4. Our patient presented with limb weakness, ataxia and visual loss in association with primary infertility due to ovarian failure. Her presentation was different from her younger sister in that, she did not have seizure or dementia4.\n\nThe diagnostic workup includes MR imaging of the central nervous system which demonstrates diffuse and cystic degenerative loss of the deep cortical white matter and U-fibers. The grey matter is preserved3,5,6. This is due to a mutation in EIF2B which causes impairment of protein synthesis under conditions of cellular stress like infection, trauma, intense emotions and surgery. The genetic tests confirm the diagnosis6. Those females who live into adulthood develop ovarian failure2,4,6. Interestingly, according to our literature search, primary testicular failure has not been described in any reported case of affected males .\n\nTreatment is largely palliative and preventive. This may include avoidance of stressors, vaccination, anti epileptic drugs and hormonal replacement therapy in affected females4,5,7.\n\n\nConclusions\n\nVWM is a rare but potentially serious disease. In adult females, premature ovarian failure is characteristic. There is no effective treatment.\n\n\nConsent\n\nWritten informed consent for publication of her clinical details and/or clinical images was obtained from the patient.", "appendix": "Competing interests\n\n\n\nThere are no competing interests to declare.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nvan der Knaap MS, Barth PG, Gabreëls FJ, et al.: A new leukoencephalopathy with vanishing white matter. Neurology. 1997; 48(4): 845–55. PubMed Abstract \| Publisher Full Text\n\nSchiffmann R, Moller JR, Trapp BD, et al.: Childhood ataxia with diffuse central nervous system hypomyelination. Ann Neurol. 1994; 35(3): 331–40. PubMed Abstract \| Publisher Full Text\n\nPronk JC, van Kollenburg B, Scheper GC, et al.: Vanishing white matter disease: A review with focus on its genetics. Ment Retard Dev Disabil Res Rev. 2006; 12(2): 123–8. PubMed Abstract \| Publisher Full Text\n\nKhan A, Humayun M, Ayub M, et al.: Vanishing White Matter (VWM) Disease presenting As Neuro-Ovarian Failure. J Coll Physicians Surg Pak. 2017; 27(3): S41–2. PubMed Abstract\n\nLabauge P, Horzinski L, Ayrignac X, et al.: Natural history of adult-onset eIF2B-related disorders: A multi-centric survey of 16 cases. Brain. 2009; 132(Pt 8): 2161–9. PubMed Abstract \| Publisher Full Text\n\nHorzinski L, Huyghe A, Cardoso MC, et al.: Eukaryotic initiation factor 2B (eIF2B) GEF activity as a diagnostic tool for EIF2B-related disorders. PLoS One. 2009; 4(12): e8318. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTurón-Viñas E, Pineda M, Cusí V, et al.: Vanishing white matter disease in a Spanish population. J Cent NervSyst Dis. 2014; 6: 59–68. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "30650", "date": "22 Mar 2018", "name": "Jeremy Chataway", "expertise": [ "Reviewer Expertise Neurology - white matter 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\nThis report look at Vanishing White Matter disease (VMD), part of the genetically inherited leukoencephalopathies, a rare group of neurological disorders, of which X-linked adrenoleukodystrophy is the most common (1/17,000). VMD is an autosomal recessive disorder characterised by progressive neurological impairment and white matter cystic degeneration. It generally presents in childhood but adult cases are well reported.\n\nHere a 30 year old woman reports a short duration of weakness, ataxia and primary infertility. An older sister also has VMD (there are siblings in total).\n\nIn females there can be ovarian failure, which is a useful diagnostic feature, as is shown here (so it can be described as an ovarioleukodystrophy). The periventricular white matter shows the same signal intensity as CSF on FLAIR/T2 weighted imaging. Mutations occur in any of the 5 genes that code for translation initiation factor EIF2B (EIF2B1 – EIF2B5), here is EIF2B. Sometimes deterioration is triggered by physical or emotional trauma.\nThis is a useful addition to the literature of a very rare group of conditions.\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": "32796", "date": "12 Apr 2018", "name": "Bernard Corenblum", "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\nPremature menopause is a term no longer accepted. Premature ovarian insufficiency is used now.\nThe endocrine aspect of this case is the hypogonadism due to ovarian failure. No speculation is given as to the association, but perhaps none is known.\nShe presented at age 30, older than most, including her sister, yet was sexually immature in development.\nThere should be more detail on the reproductive history: onset of menarche, cycle history, when did this change, etc. This phenotype appears to be a spectrum with this case later in onset than usual, so more details are needed to extend the data base on this clinical association.\nIs there any relationship with adrenal insufficiency?\n\nIs the background of the case’s history and progression described in sufficient detail? No\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? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "3592", "date": "23 Apr 2018", "name": "Abidullah Khan", "role": "Author Response", "response": "We thank Dr.Bernard Corenblum for his precious time in reviewing our manuscript. The points raised by him are praise worthy and we did our best to address his concerns. The points addressed in the light of his recommendation will definitely improve the quality of our article. The term 'premature menopause' has been rephrased as 'premature ovarian insufficiency'. More details have been provided regarding her reproductive history including onset of menarche, cycle history and the time they changed. There is no association with adrenal failure. Association of adrenal failure with leukodystrophy is a second well defined entity called as 'adreno-leukodystrophy'. Thanks again." } ] } ]	1	https://f1000research.com/articles/7-158
https://f1000research.com/articles/7-479/v1	20 Apr 18	{ "type": "Software Tool Article", "title": "ShinyDiversity - Understanding Alpha and Beta Diversity through Interactive Visualizations", "authors": [ "Eric T. Leung", "Roshan Noronha", "Ali Mirza", "Reva Shenwai", "Asterios Mpatziakas", "Roshan Noronha", "Ali Mirza", "Reva Shenwai", "Asterios Mpatziakas" ], "abstract": "In the past few decades, ecologists have developed many diversity indices to describe within and between sample diversity. Consequently, it can be difficult to determine which index to choose and how the distribution of microbial communities affect these indices. We've developed an interactive application, ShinyDiversity, that dynamically visualizes different alpha or beta diversity indices. In enabling users to select and simultaneously visualize different indices, our application aims to facilitate understanding of how the microbial data affects selected indices.", "keywords": [ "shiny", "alpha diversity", "beta diversity", "interactive web application", "R", "microbiome" ], "content": "Introduction\n\nMicrobial survey studies (i.e. microbiome survey analysis) use alpha and beta diversity indices to estimate within and between sample diversity. Alpha diversity is the diversity in a single sample site (e.g. human gut) and beta diversity describes the difference in diversity between those sites1 (e.g. different regions of the body). With a variety of alpha and beta diversity indices available, it can be difficult to determine which index to choose.\n\nPreviously developed user-friendly HTML web applications such as Microbiome Analyst2 and Dynamic Assessment of Microbial Ecology (DAME)3 allow users to visualize alpha and beta diversity. However, these tools do not address and explore how the different alpha and beta diversity indices impact their results. In this regard, we’ve developed an interactive user-friendly application that utilizes real data to dynamically visualize different alpha or beta diversity indices (Figure 1). The user is able to see how the distribution, normalization, and datasets alter the resulting diversity indices. Ultimately, this leads to an intuitive understanding of how these different diversity indices affect the data. The majority of the tool’s development was undertaken as part of the hackseq genomics hackathon in Vancouver, BC.\n\nThe homepage includes a brief description of the project, how to run the application locally, and the motivation behind the project.\n\n\nMethods\n\nShinyDiversity is an interactive HTML web application that utilizes the shiny (version 1.5.5.872) R package4. The application allows users to interactively visualize both alpha and beta diversity of multiple datasets. All diversity plots are generated using the phyloseq (version 1.16.2) R package which conveniently allows for phylogenetic analysis and visualization of microbial communities and provides 44 supported distance methods5. The underlying data used for calculations is an operational taxonomic unit (OTU) abundance table. An OTU abundance table is a matrix where the rows represent the various taxa and the columns are different samples. The table values are the counts of how often those taxa are observed.\n\nSystem requirements are computers that can successfully install Bioconductor (Release 3.6) and R (≥ 3.4.0).\n\nOur application utilizes two built-in datasets from phyloseq (version 1.16.2): GlobalPatterns and esophagus. GlobalPatterns is a dataset composed of nine different sample types obtained from areas ranging from freshwater to the human gut6. The esophagus dataset is a small example dataset of three samples of a human esophageal community, with one sample from each of the three subjects7. In addition to these two datasets, we created a third dataset GP3, which is a subset of the GlobalPatterns dataset. The following R code generates this dataset.\n\n\n\nGP3 only includes human feces, skin, and tongue samples and was created for easier visualization of multiple sample groups.\n\n\nUse cases\n\nThe alpha diversity page (Figure 2) currently gives users the option to visualize up to five different alpha diversity indices: Abundance Coverage-based Estimator (ACE), Shannon, Simpson, Inverse Simpson, and Fisher. The application dynamically produces side by side comparisons of the original data and any indices selected by the user. The side by side comparison allows the user to compare and contrast their selected indices.\n\nHere is an example with the GlobalPatterns dataset and three plots for alpha diversity indices: observed taxa (i.e. number of different taxa), ACE, and Inverse Simpson. The dropdown box allows the user to choose different datasets.\n\nIt was also important for users to have a top level and individual sample view of their data in order to quickly identify interesting features (Figure 3). The alpha diversity page features a heat map displaying the frequency count of each sample in the dataset. A barplot right beside the heat map shows the intensity pattern for a single sample, which provides a quick way to identify and focus on interesting samples.\n\nThe heat map gives a cursory view of the similarities and differences between samples. The bar graph shows an individual sample where the x-axis are the taxa and the y-axis are the taxa counts. The slider allows the user to move through the different samples to change the bar plot. These plots are shown at the bottom of the alpha diversity page.\n\nLastly, the alpha diversity page also shows the singleton and doubleton count for each sample (Figure 4). Some of the alpha diversity indices are sensitive to singletons and doubletons, which are OTUs that appear in the data only once or twice, respectively. These rare OTUs may suggest undersampling and hence a higher, true abundance in the population8. If a OTU is found more than two times then we can be more confident that it is not a false positive.\n\nThe table shows the different samples and the number of single and double taxa.\n\nCurrently, the beta diversity page (Figure 5 and Figure 6) allows users to dynamically visualize and compare two groups of the most common beta diversity indices: 1) non-phylogenetic distance indices - Euclidean, Bray-Curtis, Jaccard and 2) phylogenetic distance indices - unweighted UniFrac and weighted UniFrac. All distance indices were visualized with principal coordinate analysis (PCoA) plots, which have two principal coordinates that explain the greatest distance between samples. The dataset used to visualize beta diversity is the GP3 normalized dataset. The dataset is normalized by rarefying, a resampling method9, to the sample with the smallest library size (N = 100,187). Users have the option to rarefy the samples to any library size below 100,187. This option allows users to visualize how rarefying affects beta diversity.\n\nThe Euclidean, Bray-Curtis, and Jaccard distances are plotted. The slider will rarefy the GP3 dataset to the specified library size.\n\nThe indices plotted are the unweighted UniFrac and weighted UniFrac.\n\n\nConclusions and future work\n\nEcologists have spent decades developing these diversity indices, each having their own assumptions and use cases. Current software tools make it easy to calculate many of these indices without making the strengths and weaknesses of each clear. ShinyDiversity facilitates an exploration and understanding of alpha and beta diversity indices on microbiome data. This understanding is developed by enabling users to compare and contrast the visual differences in the plotted indices on their data.\n\nOur software is the first step in making these indices more understandable. Future work includes allowing users to input an abundance table for both the genus and OTU taxa level, use other normalization techniques (i.e. scaling), and select the degree of sparsity and dispersion. Additionally, we plan to include more diversity indices and options for users to change the distribution of selected samples. This will enable users to observe how each diversity index is influenced by sample distribution, providing a deeper understanding of diversity indices.\n\n\nSoftware availability\n\nShinyDiversity website https://erictleung.shinyapps.io/shinydiversity\n\nLatest source code: https://github.com/erictleung/shinydiversity\n\nArchived source code as at time of publication: http://dx.doi.org/10.5281/zenodo.1188304 (Leung et al., 2018)\n\nSoftware license: GNU General Public License (GPL) Version 3.0", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nEric Leung was supported by the National Library Of Medicine of the National Institutes of Health under Award Number T15LM007088. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.\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’d like to thank Akiff Manji and Varun Srinivasan for their ideas and contributions to the software’s code. We’d also like to thank the hackseq (http://www.hackseq.com/) organizing committee for giving the authors the unique opportunity to work together.\n\n\nReferences\n\nFinotello F, Mastrorilli E, Di Camillo B: Measuring the diversity of the human microbiota with targeted next-generation sequencing. Brief Bioinform. 2016; pii: bbw119. PubMed Abstract \| Publisher Full Text\n\nDhariwal A, Chong J, Habib S, et al.: MicrobiomeAnalyst: a web-based tool for comprehensive statistical, visual and meta-analysis of microbiome data. Nucleic Acids Res. 2017; 45(W1): W180–W188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiccolo BD, Wankhade UD, Chintapalli SV, et al.: Dynamic assessment of microbial ecology (DAME): a web app for interactive analysis and visualization of microbial sequencing data. Bioinformatics. 2018; 34(6): 1050–1052. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang W, Cheng J, Allaire JJ, et al.: shiny: Web Application Framework for R. 2017. Reference Source\n\nMcMurdie PJ, Holmes S: phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One. 2013; 8(4): e61217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaporaso JG, Lauber CL, Walters WA, et al.: Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A. 2011; 108 Suppl 1: 4516–4522. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPei Z, Bini EJ, Yang L, et al.: Bacterial biota in the human distal esophagus. Proc Natl Acad Sci U S A. 2004; 101(12): 4250–4255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoddington JA, Agnarsson I, Miller JA, et al.: Undersampling bias: the null hypothesis for singleton species in tropical arthropod surveys. J Anim Ecol. 2009; 78(3): 573–584. PubMed Abstract \| Publisher Full Text\n\nHughes JB, Hellmann JJ: The application of rarefaction techniques to molecular inventories of microbial diversity. Methods Enzymol. 2005; 397: 292–308. PubMed Abstract \| Publisher Full Text" }	[ { "id": "33354", "date": "03 May 2018", "name": "Jianguo Xia", "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\nLeung et al described a software tool for visual analysis of microbiome alpha and beta diversity. The motivations are to be user-friendly and to allow users to explore how different alpha and beta diversity indices impact their results. The tool is developed using Shiny and Phyloseq R package\nTheir solution is to put the graphical outputs side-by-side to facilitate comparisons. The benefits of such arrangement is very clear. However, it is important to realize that real sample size are often very large, making such arrangement not practical (esp. for alpha diversity). I also appreciate the table showing singletons and doubletons.\n\nMain limitations: I cannot find a way to upload data! Without this, it is not a tool yet, not even mention user friendliness, as it cannot be used at the moment. I would strongly urge authors to implement this very basic feature.\n\nMinor: 1) It appears that the tool should be installed from within the RStudio. If I do it from R terminal, there will be errors; 2) Fig 3 should be normalized for more informative view.\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": "33341", "date": "16 May 2018", "name": "Richard E. Isaacson", "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 advent of microbial ecologic studies using 16S rRNA gene analysis has opened the field of microbial diversity to a wide range of new investigators. Most investigations include ecologic analyses of microbial diversity using a range of different diversity indices. The authors of the ShinyDiversity have developed a new software package that can aid the investigator in these analyses. Appropriate to its use is the interactive nature of the package. ShinyDiversity should be of value to many investigators in the field. Users, however, should be aware that interpretation of results needs to be in the context of the broad assumptions built in to the diversity indices. In particular, those using this tool probably are comparing ecologic environments and that identifying differences in diversity can be used to imply differences in microbial community makeup based on some differences in the environment. However, minor differences can occur as the result of defined microbial differences that may or may not grossly affect the diversity indices. Thus, interpretation of the result must go beyond the integration of large data sets into indices but need to take in to account the true compositionally differences. I believe that the authors do understand 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? Partly\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/7-479
https://f1000research.com/articles/7-138/v1	01 Feb 18	{ "type": "Research Article", "title": "Classification of processes involved in sharing individual participant data from clinical trials", "authors": [ "Christian Ohmann", "Steve Canham", "Rita Banzi", "Wolfgang Kuchinke", "Serena Battaglia", "Steve Canham", "Rita Banzi", "Wolfgang Kuchinke", "Serena Battaglia" ], "abstract": "Background: In recent years, a cultural change in the handling of data from research has resulted in the strong promotion of a culture of openness and increased sharing of data. In the area of clinical trials, sharing of individual participant data involves a complex set of processes and the interaction of many actors and actions. Individual services/tools to support data sharing are available, but what is missing is a detailed, structured and comprehensive list of processes/subprocesses involved and tools/services needed. Methods: Principles and recommendations from a published data sharing consensus document are analysed in detail by a small expert group. Processes/subprocesses involved in data sharing are identified and linked to actors and possible services/tools. Definitions are adapted from the business process model and notation (BPMN) and applied in the analysis. Results: A detailed and comprehensive list of individual processes/subprocesses involved in data sharing, structured according to 9 main processes, is provided. Possible tools/services to support these processes/subprocesses are identified and grouped according to major type of support. Conclusions: The list of individual processes/subprocesses and tools/services identified is a first step towards development of a generic framework or architecture for sharing of data from clinical trials. Such a framework is strongly needed to give an overview of how various actors, research processes and services could form an interoperable system for data sharing.", "keywords": [ "clinical trial", "data sharing", "individual participant data (IPD)", "process", "business process model", "generic framework" ], "content": "Abbreviations\n\nAAI, Authentication and Authorisation Infrastructure; API, Application Programming Interface; ATT, The Open Science and Research Initiative; BPMN, Business Process Model and Notation; BRIDG, Biomedical Research Integrated Domain Group; CDISC CDASH, Clinical Data Interchange Standards Consortium - Clinical Data Acquisition Standards Harmonization; CDISC ODM, Clinical Data Interchange Standards Consortium - Operational Data Model CDISC SDM, Clinical Data Interchange Standards Consortium - Study Design Model; COMET, Core Outcome Measures in Effectiveness Trials; CORBEL, Coordinated Research Infrastructures Building Enduring Life-science Services; CRUK, Cancer Research UK; DOI, Digital Object Identifier; ECRIN, European Clinical Research Infrastructure Network; ID, Identity; IPD, Individual Participant Data; IT, Information Technology; MRC, Medical Research Council (UK); PCROM, Primary Care Research Object Model; QA, Quality Assurance; UK, United Kingdom; UKCRC, UK Clinical Research Consortium; US, United States; WHO, World Health Organization\n\n\nIntroduction\n\nIn recent years, a cultural change in the handling of research data has resulted in the strong promotion of a culture of openness and increased sharing of data. Many organisations, initiatives and projects have expressed their commitment to support open scientific research. This move has been extended also to clinical trials. Today, the results of clinical trials are more and more considered as a public good, and access to the individual participant data (IPD) generated by those trials is seen as part of a fundamental right to health data (see Research Councils UK principles on data policy).\n\nTo support data sharing in clinical trials, several organisations have developed generic principles, guidance and practical recommendations for implementation in recent years (e.g. the Institute of Medicine report in the US1, the Nordic Trial Alliance Working Group on Transparency and Registration for the Nordic countries2, the good practice principles for sharing IPD from publicly funded trials by MRC, UKCRC, CRUK and Wellcome, in the UK3,4, or the guide to publishing and sharing sensitive data for Australia5). Within the EU Horizon 2020 funded project CORBEL (Coordinated Research Infrastructures Building Enduring Life-science Services) and coordinated by the European Clinical Research Infrastructure Network (ECRIN), an interdisciplinary and international stakeholder taskforce reached a detailed consensus on principles and recommendations for data sharing of clinical trial data6. That document was taken as the starting point for the current paper.\n\nData sharing of IPD from clinical trials involves a complex set of processes and the interaction of many actors and actions. Some documentary support is available, (e.g. templates for data sharing plans, data transfer and data use agreements), but this is scattered and thus not always easy to find. In addition, although some IT-tools and services are available to give support for individual tasks in the process of data sharing (e.g. de-identification service for datasets; see Electronic Health Information Laboratory page on de-identification software) or an ID-generation service for study objects), these are again difficult to discover and their quality is not easy to explore. An additional aspect of complexity stems from the very heterogeneous set of repositories that are available for storage of IPD (see Registry of Research Data Repositories). There are general scientific repositories, repositories dedicated specifically to clinical research, repositories specialised in storing data related to a specific disease area and institution-specific repositories. In summary, although fragments of infrastructure are available to support sharing of IPD from clinical trials, the various services and tools are scattered and a global vision of how all these components should interact and interoperate does not currently exist.\n\nWhat is still missing is a generic framework or architecture for data sharing that could be used for modelling, describing, and designing operations, data requirements, IT-systems and technological solutions (see Open Group TOGAF® framework). Such a framework would link structural concepts (e.g. actors) with behavioural concepts (e.g. processes linked to services) giving an overview of how actors, processes and services interact to form a system for data sharing of IPD. Due to its complexity with many different processes and actors, such a framework is not available at the moment. As a first step in creating such a framework, in this paper we provide a systematic, structured and comprehensive list of processes/ subprocesses linked to data sharing derived from our CORBEL consensus document.\n\n\nMethods\n\nRecommendations and principles from the data sharing consensus document were analysed in detail and individual processes/subprocesses identified and linked to actors and possible services/tools by a small group of experts (CO, SC, RB, WK, SB). The consensus document covers all stages of the data sharing life cycle and is highly structured, with 7 main topics, 10 principles assigned to these topics and 50 specific recommendations, making the analysis process relatively straightforward6. The specification of processes/subprocesses, actors and services/tools was agreed between the experts in telephone conferences and by written communication, and summarized in a table with listings.\n\nIn the next step, possible services/tools associated with single processes/subprocesses were analysed and grouped according to different types of support, preserving reference to the processes/subprocesses specified in the first step.\n\nThe following definitions were adapted from the business process model and notation (BPMN) and applied to our analysis (see Object Management Group page, 7):\n\nProcess: A sequence or flow of activities in an organization with the objective of carrying out work (see Object Management Group page).\n\nIn this study, processes may relate to different organisations and business goals, e.g. the various activities of the data generators, data storage managers and secondary users all represent different business processes, operating at different times by different actors.\n\nSubprocess: A process that is included within another process (see Object Management Group page)\n\nActor: Some person or organization taking part in day-to-day business activity (see Object Management Group page)\n\nActors are belonging to or have a relationship with the clinical trial arena. Actors include: investigators, trial unit heads, QA-staff, senior data management and IT-staff, trial unit operational managers, statisticians, sponsors, trial management team, specialist agencies, repository managers, analysis environment providers, secondary users of data, data use advisory panel, research infrastructures, journal publishers, patient representatives, and funders. Definitions of actors have been taken from the glossary in the consensus document6 and some from the CDISC-glossary.\n\nService: A service is a functional business entity that fulfils a particular requirement (see Open Science and Research framework)\n\nServices/tools may be relatively non-technical (e.g. providing information, example materials, template policies and procedures, assessment criteria, metadata, and infrastructure specifications) or technical, i.e. information technology based. The technology required may be conventional (e.g. webpages, web-based information systems) and already available (though would need normally need specific organisation and application). Other services/tools may require specialist software development (e.g. development of an analysis environment, developing systems to support metadata repositories).\n\nSubservice: A subservice is a special case of a service (see Open Science and Research framework)\n\nTo keep things as simple as possible, processes were structured according to the main activities within data sharing of IPD and then further differentiated with respect to subprocesses. For every process the involved actors and possible tools/services are linked.\n\nFor graphical illustration, the BPMN approach was used. In BPMN, a process is depicted as a graph of flow elements, which are a set of activities, events, gateways, and sequence flow that adhere to a finite execution semantics. The usual BMBP notation and symbols were taken (event, activity, gateway, connections, swim lane) (see Object Management Group page). In this publication, BPMN is used only to give a high-level overview on the relation between the main processes.\n\n\nResults\n\nFrom the analysis of the consensus document 9 main processes involved in data sharing of IPD were identified:\n\n1. Preparation for data sharing, in general\n\n2. Plan for data sharing, in the context of a specific trial\n\n3. Preparation of data for sharing, after data collected\n\n4. Transferring data objects to an external repository\n\n5. Repository data and access management\n\n6. Access to individual participant data and associated data objects\n\n7. Discovering the data objects available\n\n8. Publishing results of re-use\n\n9. Monitoring data sharing\n\nProcess 1 to 5 can be summarized under the heading “Data preparation and storage”, the processes 6-9 under the heading “Data request and secondary analysis”. The relationship between the main processes is presented in Figure 1.\n\nThe main processes were structured further into more detailed processes/subprocesses and linked to actors involved and possible services/tools. As result a detailed and comprehensive list of individual processes/subprocesses involved in data sharing is given in Table 1.\n\n1Data objects: any discrete packages of data in an electronic form – whether that data is textual, numerical, a structured dataset, an image, film clip, (etc.) in form. They are each a file, as that term is used within computer systems, and are named, at least within their source file system. In the context of clinical research and data sharing, data objects can include electronic forms of protocols, journal papers, patient consent forms, analysis plans, and any other documents associated with the study, as well as datasets representing different portions and types of the data generated, and the metadata describing that data.\n\n2Authentication: The process of ensuring that a person or system that is trying to access a system is who they say (it says) they are. With a person, authentication is by provision of one or more of something only they should know (e.g. a password), or should have (e.g. a card or fob), or can show (e.g. fingerprint, iris pattern). With a system it is more often by provision of a secret token (in effect a machine password), often derived from public key cryptography.\n\nAuthorisation: The process of giving an authenticated entity the rights to access particular subsets of data and/or to carry out particular functions within a system. It is usually carried out by assigning user entities to roles and to groups that together define the access allowed.\n\nIn Table 2, possible services/tools associated with processes are grouped according to major types of support, preserving reference to the processes/subprocesses. As the table illustrates, these tools and services fall into 6 (overlapping) categories:\n\n1. Providing general background material\n\n2. Locator services (for resources for data sharing, and / or to support data standards)\n\n3. Example documents and templates\n\n4. Services (e.g. to de-identify data, assign IDs, provide metadata, evaluate repositories)\n\n5. Frameworks and guidance (e.g. metadata schemas, citation systems, checklists)\n\n6. Tools (IT based, e.g. APIs to harvest repository contents, tools to assign metadata)\n\n\nDiscussion\n\nWithin the framework of the EU H2020 funded project CORBEL major issues associated with sharing of IPD were investigated and a consensus document on providing access to IPD from clinical trials was developed, using a broad interdisciplinary approach6. The taskforce reached consensus on 10 principles and 50 recommendations, representing the fundamental requirements of any framework used for the sharing of clinical trials data. To support the adoption of the recommendations, adequate tools and services are needed to promote and support data sharing and re-use amongst researchers, adequately inform trial participants and protect their rights, and provide effective and efficient systems for preparing, storing, and accessing data. As a first step on the way to inventory existing tools/services, their quality and applicability for data sharing, a systematic analysis of processes and actors involved in data sharing was performed. The work done resulted in a systematic, structured and comprehensive list of processes/subprocesses that need to be supported to make data sharing a reality in the future. It is basic work against which existing tools/services can be mapped, and gaps, where new tools/services are needed, can be identified.\n\nIn the context of this work, we explored the possibility of generating a generic framework for the sharing of IPD from clinical trials. As an example we considered the Framework for Open Science and Research by ATT (see Open Science and Research framework). This framework provides a general description of the desired architecture in a domain of open science. The framework configures and defines the key structural elements of the overall solution. It gives an overview of how various actors, research processes and services – including data, data structures, actors, roles and IT-systems – could form an interoperable system in the ‘target’ open state. The Enterprise Architecture (EA) approach is used, modelling, describing and designing operations, data requirements, IT-systems and technical solutions in accordance with a common model. The work done in developing a framework for open science and research could be of major relevance for a similar model in the area of data sharing. At this stage, of trying to basically structure processes/subprocesses involved in data sharing, it was seen as too early to develop a generic framework. It may, however, be that this approach is taken up again when the basic work has been done and the components for such a framework have been identified.\n\nNevertheless, we thought it useful to use a standardised terminology and notation for describing basic processes in data sharing. This will simplify the extension to a more generic and comprehensive framework at a later stage. As one approach, business modelling has been applied successfully in the health and health research area. It has been used, for example, to perform a requirements analysis of the barriers to conducting research linking of primary care, genetic and cancer data7, to model the complexity of health and associated data flow in asthma8 and to provide a generic architecture for a type 2 diabetes mellitus care system9. We decided not to apply the full spectrum of business process modelling (BPMN), but to use only basic elements to give a notational and terminological basis for further work. This does not imply, however, that the application of the full spectrum of BPMN techniques is a necessary step in developing an overall framework. More work is needed to explore the suitability and benefit of BPMN for a generic framework for data sharing.\n\nDifferent models for clinical trials and clinical trials workflow already exist, such as the domain analysis model BRIDG10, the study design model CDISC SDM11 and the primary care information model PCROM12. Any framework or model for data sharing needs to map or reference these clinical trial models, though none currently include the secondary use of data after the trial has completed. Although clinical trial processes and data sharing processes are distinct, they are clearly linked, and any models need to incorporate those linkages. As a consequence, developing a generic framework or architecture for data sharing needs much more work and is not covered in this paper.\n\nMany of the services/tools identified in this paper are non-technical but nevertheless may be of major importance, especially for data generators and data requestors. This includes templates/examples, checklists and guidance. For some of the processes specified in this paper IT-tools and services already exist and can be applied (e.g. de-identification tools and services, see Electronic Health Information Laboratory page on de-identification software), others are under development or need improvement (e.g. metadata repository for identifying clinical trial objects, 13). The next step is to perform a scan on the availability and suitability of services/tools for data sharing based on this work, with the involvement of stakeholders. We will summarize this information in a separate report.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project has received funding from the European Union's Horizon 2020 research and innovation programme (CORBEL, under grant agreement n° 654248).\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 wish to thank Mihaela Matei (ECRIN) for support with legal issues.\n\n\nReferences\n\nInstitute of Medicine: Sharing Clinical Trial Data, Maximizing Benefits, Minimizing Risk. Washington, DC: National Academies Press (US). 2015. Publisher Full Text\n\nSkoog M, Saarimäki JM, Gluud C, et al.: Report on Transparency and Registration in Clinical Research in the Nordic countries. Nordic Trial Alliance Working Group 6 on Transparency and Registration. 2015; accessed 15/01/2018. Reference Source\n\nTudur Smith C, Hopkins C, Sydes M, et al.: Good Practice Principles for Sharing Individual Participant Data from Publicly Funded Clinical Trials. 2015; accessed 15/01/2018. Reference Source\n\nTudur Smith C, Hopkins C, Sydes MR, et al.: How should individual participant data (IPD) from publicly funded clinical trials be shared? BMC Med. 2015; 13: 298. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nANDS guide: Publishing and sharing sensitive data. Australian National Data Service. 2017; accessed 15/01/2018. Reference Source\n\nOhmann C, Banzi R, Canham S, et al.: Sharing and reuse of individual participant data from clinical trials: principles and recommendations. BMJ Open. 2017; 7(12): e018647. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Lusignan S, Krause P, Michalakidis G, et al.: Business Process Modelling is an Essential Part of a Requirements Analysis. Contribution of EFMI Primary Care Working Group. Yearb Med Inform. 2012; 7: 34–43. PubMed Abstract\n\nLiyanage H, Luzi D, De Lusignan S, et al.: Accessible Modelling of Complexity in Health (AMoCH) and associated data flows: asthma as an exemplar. J Innov Health Inform. 2016; 23(1): 863. PubMed Abstract \| Publisher Full Text\n\nUribe GA, Blobel B, López DM, et al.: A generic architecture for an adaptive, interoperable and intelligent type 2 diabetes mellitus care system. Stud Health Technol Inform. 2015; 211: 121–131. PubMed Abstract \| Publisher Full Text\n\nBiomedical Research Integrated Domain Group (BRIDG). Release 3.1 Comprehensive Domain Analysis Model Static Elements Report. Generated from Enterprise Architect, accessed 15/01/2018. 2012. Reference Source\n\nClinical Data Interchange Standards Consortium (CDISC): CDISC Study Design Model in XML (SDM-XML). Release version 1.0, 2008–2011, accessed 15/01/2018. Reference Source\n\nKuchinke W, Karakoyun T, Ohmann C, et al.: Extension of the primary care research object model (PCROM) as clinical research information model (CRIM) for the \"learning healthcare system\". BMC Med Inform Decis Mak. 2014; 14: 118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoldacre B, Gray J: OpenTrials: towards a collaborative open database of all available information on all clinical trials. Trials. 2016; 17: 164. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "31016", "date": "01 Mar 2018", "name": "Florian Naudet", "expertise": [ "Reviewer Expertise Meta-research" ], "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 Classification of processes involved in sharing individual participant data from clinical trials by Ohmann C, Canham S, Banzi R, Kuchinke W and Battaglia S1 is more than useful for all stakeholders interested in data sharing. It must be accepted with, in my opinion, a few (and minor) edits.\n\nIn my experience as a researcher interested in the impact of data sharing policies2, I have identified that a major practical barrier to implementation of full data sharing of randomised controlled trials was the great heterogeneity across different trial groups: \"getting prepared and preplanning for data sharing still seems to be a challenge for many trial groups; data sharing proved to be novel for some authors who were unsure how to proceed\". Therefore the description and classification of processes involved in sharing IPD from clinical trials will surely helps all stakeholders to get prepared. It is welcome and this manuscript will be very useful.\n\nI have a few suggestions that may help to write it better. Please note that I'm not an expert in qualitative research. Therefore these are only suggestion that I don't want to enforce strongly.\n\nFirst, as it is presented as a research paper and because it is very qualitative by nature, I would suggest to use, or better adapt the reporting guidelines for qualitative research3 to this specific paper as most points won't directly apply since the study presented is not a typical qualitative research.\n\nMore specifically, I would welcome more details on authors in the main text:\n\n- Who are they? Were they from different background (e.g. data managers, statisticians, trialists, patients, etc..., Master degree, MD, PhD, PharmD... etc.). Please clearly state that they were involved in the initial initiative that was used for this paper4. Please also detail how it could have affected their judgement.\n\n- What is their background for conducting such a qualitative synthesis?\n\n- Was there a protocol registered for this analysis?\n\nPlease specify why the processes were derived from only one initiative4 and not from a systematic assessment of other papers/initiatives. Any limitations of the initial paper should be discussed here. The process of analysis should be made as transparent as possible. How the different authors were involved in the process? Were there some leaders during the phone meetings? Were verbatim from written correspondence used? Was there a good agreement between expert (for what parts the agreement was less good ?)? The researchers’ own position should also clearly be stated. A critical examination of their own role, possible bias, and influence on the research would be welcome.\n\nI have also identified very practical points that could be addressed in a new version of the manuscript:\n\n- In my very practical experience2, figure 1 could be overly simple for being accurate. I think that one important point was missed. Adoption of data sharing in biomedical research not only implies to provide and re-use the data. It implies to adopt a collaborative approach. It means that when one want to re-use the data of another team, one sometimes must directly contact the other team to have information and to have the data in the appropriate format. Sharing data for a re-analysis of safety outcomes involves sharing the cases report forms while re-using data for some IPD meta-analysis may only rely on sharing data at a later analytical stage (e.g. analysable data). This implies that step 3 is very linked with step 6. I think that the figure will be better (if it is not too complex) by adding such kind of relationship.\n\n- Table 1, section 1.1.1 / 2.3.1: patients are an important actors/leverages and must be involved in my opinion in these aspects ;\n\n- Table 1, in general avoid abbreviations such as \"SOP\" in 1.3 ;\n\n- Table 1, section 2 and 3.1: Ethic committees have a strong role to play at all these parts. They have, in my opinion to judge wether the de-identification plan is adapted to the specific study ;\n\n- Table 1, section 3.2.1: data manager and statisticians must ensure that the code that will be shared works for the de identified data sets. Practical finding from my experience (in one case, de-identification was made after the analysis and labels were different between the two datasets : therefore the shared code didn't worked).\n\n- Table 1, section 4.1.1: this should be explored before in my opinion (at step 3), when one decide of the data sharing plan.\n\n- Table 2 very interesting, but I would suggest to add an hyperlink to some concrete examples when possible in section 3.\n\nIn general the tables should be checked for majuscule and minuscule: eg. table 2, section 3 \"during\" must be During.\n\nA last suggestion would be to add more practical information for clinicians and to cite the ICJME recommandations.\n\nIt is again a very great manuscript and I hope that these comment will be able to improve it.\n\nI'm not competent to review the English, and please excuse my English.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3596", "date": "20 Apr 2018", "name": "Christian Ohmann", "role": "Author Response", "response": "Response to the reviewer in bold and italics The manuscript Classification of processes involved in sharing individual participant data from clinical trials by Ohmann C, Canham S, Banzi R, Kuchinke W and Battaglia S1 is more than useful for all stakeholders interested in data sharing. It must be accepted with, in my opinion, a few (and minor) edits. In my experience as a researcher interested in the impact of data sharing policies2, I have identified that a major practical barrier to implementation of full data sharing of randomised controlled trials was the great heterogeneity across different trial groups: \"getting prepared and preplanning for data sharing still seems to be a challenge for many trial groups; data sharing proved to be novel for some authors who were unsure how to proceed\". Therefore the description and classification of processes involved in sharing IPD from clinical trials will surely helps all stakeholders to get prepared. It is welcome and this manuscript will be very useful. I have a few suggestions that may help to write it better. Please note that I'm not an expert in qualitative research. Therefore these are only suggestion that I don't want to enforce strongly. First, as it is presented as a research paper and because it is very qualitative by nature, I would suggest to use, or better adapt the reporting guidelines for qualitative research3 to this specific paper as most points won't directly apply since the study presented is not a typical qualitative research. More specifically, I would welcome more details on authors in the main text: - Who are they? Were they from different background (e.g. data managers, statisticians, trialists, patients, etc..., Master degree, MD, PhD, PharmD... etc.). Please clearly state that they were involved in the initial initiative that was used for this paper4. Please also detail how it could have affected their judgement. - What is their background for conducting such a qualitative synthesis? - Was there a protocol registered for this analysis? Please specify why the processes were derived from only one initiative4 and not from a systematic assessment of other papers/initiatives. Any limitations of the initial paper should be discussed here.The process of analysis should be made as transparent as possible. How the different authors were involved in the process? Were there some leaders during the phone meetings? Were verbatim from written correspondence used? Was there a good agreement between expert (for what parts the agreement was less good ?)? The researchers’ own position should also clearly be stated. A critical examination of their own role, possible bias, and influence on the research would be welcome. We agree with the reviewer that this paper can be classified as qualitative research, although we applied a semi-formal collaborative small group decision-making approach and not a formal methodology such as interviews or focus groups. We revised the manuscript and adapted it as much as possible to the COREQ guidelines.. However, as expected, many COREQ items are clearly not applicable. We hope this revision had improved the paper reporting. I have also identified very practical points that could be addressed in a new version of the manuscript: - In my very practical experience2, figure 1 could be overly simple for being accurate. I think that one important point was missed. Adoption of data sharing in biomedical research not only implies to provide and re-use the data. It implies to adopt a collaborative approach. It means that when one want to re-use the data of another team, one sometimes must directly contact the other team to have information and to have the data in the appropriate format. Sharing data for a re-analysis of safety outcomes involves sharing the cases report forms while re-using data for some IPD meta-analysis may only rely on sharing data at a later analytical stage (e.g. analysable data). This implies that step 3 is very linked with step 6. I think that the figure will be better (if it is not too complex) by adding such kind of relationship. According to the suggestions of the reviewer, a relation between data requester and data generator named « optional collaboration » has been added to the figure. In our consensus exercise (BMJ Open paper) we formulated the following recommendation (no. 33) : « Collaboration between data providers and secondary data users could be an added value in data sharing. However, it should not be a pre-requisite for data sharing. ». Therefore we marked the relation with « optional ». - Table 1, section 1.1.1 / 2.3.1: patients are an important actors/leverages and must be involved in my opinion in these aspects ; Added patient groups to list of actors for 1.1.1, 2.3.1 and 2.3.2 - Table 1, in general avoid abbreviations such as \"SOP\" in 1.3 ; A brief definition has been added to the glossary of at the bottom of table 1.. - Table 1, section 2 and 3.1: Ethic committees have a strong role to play at all these parts. They have, in my opinion to judge wether the de-identification plan is adapted to the specific study ; We are not sure if the exact role of ethics committees in data sharing has been clarified, though if the proposals are in the protocol and the participant information sheet (etc.) they would be scrutinised by an ethics committee. Not sure if this needs to be added explicitly as part of the workflow unless ECs are given a formal role.- Table 1, section 3.2.1: data manager and statisticians must ensure that the code that will be shared works for the de identified data sets. Practical finding from my experience (in one case, de-identification was made after the analysis and labels were different between the two datasets : therefore the shared code didn't worked). An extra subprocess has been added as 3.2.2.- Table 1, section 4.1.1: this should be explored before in my opinion (at step 3), when one decide of the data sharing plan. We are not so sure. This will never be a simple linear process, so the order in the table does not imply a similar ordering of workflow. We have changed 4.1. so that it is either a selection or a confirmation of an earlier repository selection. - Table 2 very interesting, but I would suggest to add an hyperlink to some concrete examples when possible in section 3. Table 1 and 2 were improved, taken the comments from the reviewer into consideration. In general the tables should be checked for majuscule and minuscule: eg. table 2, section 3 \"during\" must be During. Checked. A last suggestion would be to add more practical information for clinicians and to cite the ICJME recommandations. The activity of ICMJE was cited. It is again a very great manuscript and I hope that these comment will be able to improve it. I'm not competent to review the English, and please excuse my English." } ] }, { "id": "31482", "date": "19 Mar 2018", "name": "Matthew R. Sydes", "expertise": [ "Reviewer Expertise Clinical trials and clinical trial methodology" ], "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 process-orientated manuscript covers a lot of ground in some detail. I have some specific comments:\n\nMajor\n\nSection: General Comment: The process of reaching these recommendations is unclear to me. Perhaps these are opinions? I don’t think there is primary evidence to underpin them. Should there be?\n\nSection: General Comment: This is comprehensive, but also sets out a substantial burden on organisations. I wonder for what proportion of trials this work is proportionate effort.\n\nSection: General Comment: This does not address my previous concerns about recognition of effort of the original researchers or issues about self-identification by patients, but perhaps that is outside of the scope of the paper. It would helpful to remind the reader that these are key, unresolved issues and point to places where they might be considered further.\n\nModerate\n\nSection: Table 1 Text ref: \"1.2 Clarify own institution’s requirements for data sharing\" Comment: This is pretty vague. I don’t know how to use this row.\n\nSection: Table 1 Text ref: \"2.1.2 Check funder requirements for data sharing\" Comment: Which takes priority and when? 2.1.2 vs 1.2.\n\nSection: Table 1 Text ref: \"2. Plan for data sharing, in the context of a specific trial\" Comment: When should this be developed? 2.2.2 suggests before the protocol is finalised; but I suspect 2.2.2 would generally be done before 2.2.1. What is the ordering of the rows?\n\nSection: Table 1 Text ref: \"3.1 Decide upon strategy for data preparation for sharing\" Comment: 3.1.1 and 3.1.2 seem to be in the wrong order.\n\nSection: Table 1 Text ref: Section 3 or 4 Comment: Somewhere, perhaps, one should advertise the timelines for making data available. It’s unlikely to be during the trial; how long after primary analyses? Useful to manage expectations?\n\nSection: Table 1 Text ref: \"5.1 Maintain highly granular access control to IPD, that can be changed rapidly\" Comment: Changed on what basis?\n\nSection: Table 1 Text ref: \"5.5 Provide an expert advisory panel\" Comment: Is this a Data Access Committee or something different? Is there independent membership?\n\nSection: Table 1 Text ref: \"5.7 Provide data use agreement templates\" Comment: Possibly wishful thinking. Agreements are never as straightforward as one might hope. Is this a suggestion for global templates, institution templates or trial templates?\n\nSection: Table 1 Text ref: \"6.1.2 Assess the reasonableness of the request and the ability of the requesters to draw sensible conclusions\" Comment: Where is the independence in this process? Is there a duty from the sponsor and TMG to work fairly? Who judges what is reasonable?\n\nSection: Table 1 Text ref :: \"6.2.1 Repository makes appropriate request forms available on-line\" Comment: Why? This will just encourage false positive submissions. Better for applicants to talk to the trial team before getting a form, so the applicant really understands whether the data set is suitable and timely. (Very often, it really won’t be.)\n\nSection Table 1 Text refL \"7.2 Agree an ID generation scheme for data objects\" Comment: Also, what if the same dataset is given to two separate people: does this get the same ID?\n\nSection: Table 1 Text ref: \"8. Publishing results of re-use\" Comment: Who checks that the secondary use of the data is done well?\n\nSection: Table 1 Text ref: \"8. Publishing results of re-use\" Comment: What to do if there is discrepancy in findings between original and subsequent findings? Could undermine trust. Probably needs rows about “dispute” resolution.\n\nSection: Table 2 Text ref: \"2. Locator services. Locator service for data sharing resources\" Comment: Will this be a familiar term to readers? I’m not sure what it means.\n\nTrivial/Minor\n\nSection: Table 1 Comment: Would be quickly for each actor to find the role if this column was broken into separate columns, one per actor type, with the ticks for whether it is relevant.\n\nSection: Table 1 Text ref: \"7.2 Agree an ID generation scheme for data objects\" Comment: “Data objects” needs a clear definition before the table. Perhaps a Glossary with the Abbreviations?\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? No\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": [ { "c_id": "3595", "date": "20 Apr 2018", "name": "Christian Ohmann", "role": "Author Response", "response": "Response to reviewer in bold and italics This process-orientated manuscript covers a lot of ground in some detail. I have some specific comments: Major Section: GeneralComment: The process of reaching these recommendations is unclear to me. Perhaps these are opinions? I don’t think there is primary evidence to underpin them. Should there be?Principles and recommendations on data sharing were developed in the BMJ Open paper. In this study a framework based upon these principles and recommendations was proposed, characterising processes/suprocesses as well as tools/services needed for data sharing. The following methodological approach was followed. The basic concepts and definitions were adapted from the business process model and notation (BPMN) and applied to our analysis. Recommendations and principles from the data sharing consensus document were analysed in detail and individual processes/subprocesses identified and linked to actors and possible services/tools by a small group of experts (CO, SC, RB, WK, SB). The decision-making process was based on a facilitator (CO) providing initial and updated versions of the document and iterative rounds of written feedback from the team members. The process was continued until final agreement was achieved. The process took place between October 2017 and January 2018, four different versions were provided and approved in sequential order (24 November 2017, 7 and 11 December 2017, 15 January 2018). Due to the good relationship between the team members and long-term involvement in common projects, a comprehensive and detailed point of reference, the consensus document, and clear objectives with milestones and time lines, agreement could be achieved by the team without applying a normative model of decision-making. As suggested by another reviewer, this paper can be classified as qualitative research, although we applied a semi-formal collaborative small group decision-making approach and not formal methodology such as interviews or focus groups. We revised the methodological section of the manuscript and adapted it as much as possible to the COREQ guidelines for qualitative research. Section: GeneralComment: This is comprehensive, but also sets out a substantial burden on organisations. I wonder for what proportion of trials this work is proportionate effort.This is difficult to estimate. The empirical assessment of the benefit of data sharing in comparison to the effort and resources needed is an area, where much more research is needed. This issue has been explored in more detail in the BMJ Open publication but was not tackled in this paper. Section: General Comment: This does not address my previous concerns about recognition of effort of the original researchers or issues about self-identification by patients, but perhaps that is outside of the scope of the paper. It would helpful to remind the reader that these are key, unresolved issues and point to places where they might be considered further.These aspects have been discussed in detail in the BMJ open publication and are outside the scope of this paper. As suggested, readers are reminded that the points raised by the reviewer are key unsolved issues and initiatives dealing with these issues are referred to. Moderate Section: Table 1 Text ref: \"1.2 Clarify own institution’s requirements for data sharing\"Comment: This is pretty vague. I don’t know how to use this row.This was split into two subprocesses and a comment was added in the table. The order of 1.2 and 1.3 was reversed. Section: Table 1 Text ref: \"2.1.2 Check funder requirements for data sharing\"Comment: Which takes priority and when? 2.1.2 vs 1.2.Certainly a reasonable question but so far no priorities have been defined and the timely order of processes has only be lightly tackled in the figure. The work is part of ongoing research in the CORBEL project. A comment about \"clarification of legal responsibilities\" has been added in 2.1.2. Section: Table 1 Text ref: \"2. Plan for data sharing, in the context of a specific trial\"Comment: When should this be developed? 2.2.2 suggests before the protocol is finalised; but I suspect 2.2.2 would generally be done before 2.2.1. What is the ordering of the rows?Correct, the order of 2.2.1 and 2.2.2 has been reversed. Section: Table 1Text ref: \"3.1 Decide upon strategy for data preparation for sharing\"Comment: 3.1.1 and 3.1.2 seem to be in the wrong order.We have not changed that because from our viewpoint this seems to be the right order. Section: Table 1 Text ref: Section 3 or 4Comment: Somewhere, perhaps, one should advertise the timelines for making data available. It’s unlikely to be during the trial; how long after primary analyses? Useful to manage expectations?This is an important issue, which has also been discussed in the BMJ Open paper. We have included a reference to timelines in 3.1. Section: Table 1 Text ref: \"5.1 Maintain highly granular access control to IPD, that can be changed rapidly\"Comment: Changed on what basis?We removed the reference to \"rapid change\" as it seems tob e confusing. Section: Table 1 Text ref: \"5.5 Provide an expert advisory panel\"Comment: Is this a Data Access Committee or something different? Is there independent membership? The reference was changed to a Data Access Committee. We also re-organised the processes in section 5 to make them (I hope) easier to read and understand, though the content is almost exactly the same. 5.3 and 5.4 were split up into sub-processes, 5.5 – 5.7 made subprocesses of a new 5.5, and 5.6 (was 5.8) expanded to include 2 subprocesses of reporting / feedback Section: Table 1Text ref: \"5.7 Provide data use agreement templates\"Comment: Possibly wishful thinking. Agreements are never as straightforward as one might hope. Is this a suggestion for global templates, institution templates or trial templates?We agree that in practice there will be no agreed templates. Therefore we added a phrase that the templates may be starting points for negotiated, specific agreements. Section: Table 1 Text ref: \"6.1.2 Assess the reasonableness of the request and the ability of the requesters to draw sensible conclusions\"Comment: Where is the independence in this process? Is there a duty from the sponsor and TMG to work fairly? Who judges what is reasonable?Yes, a critical issue. This is the reason why we prefer data sharing via trusted repositories with defined and transparent governance. In 6.1 processes are specified for the use case of access via direct contact with the sponsor/PI. Here an independency of processes is usually not given. Section: Table 1 Text ref :: \"6.2.1 Repository makes appropriate request forms available on-line\"Comment: Why? This will just encourage false positive submissions. Better for applicants to talk to the trial team before getting a form, so the applicant really understands whether the data set is suitable and timely. (Very often, it really won’t be.)We are supporting the view that data sharing and re-use should be possible without the (mandatory) involvement of data generators. False positive submission may be reduced if the data available are fully described. According to the suggestions of another reviewer, a relation between data requester and data generator named « optional collaboration » has been added to the figure. In our consensus exercise (BMJ Open paper) we formulated the following recommendation (no. 33) : « Collaboration between data providers and secondary data users could be an added value in data sharing. However, it should not be a pre-requisite for data sharing. ». Therefore we marked the relation with « optional ». Section Table 1 Text refL \"7.2 Agree an ID generation scheme for data objects\"Comment: Also, what if the same dataset is given to two separate people: does this get the same ID?Yes, the ID is fixed with the clinical trial objects. 7.2. is now split into two related subprocesses, as is 7.3. 7.1. and 7.5 simplified by removal of subprocess. Section: Table 1 Text ref: \"8. Publishing results of re-use\"Comment: Who checks that the secondary use of the data is done well?Yes, this is a critical issue. There is no standard procedure foreseen for this. The best strategy is to make the re-analysis fully open and transparent. (see 8.1.1). In that case the scientific community (including the data generators) can check the validity of the re-analysis. Nevertheless, monitoring compliance (in general) is an open issue but not impossible. FDAA Trial Tracker is a good example of monitoring compliance to regulation in trial registry and Ben Goldacre’s group is also chasing and publishing non-compliance. Section: Table 1 Text ref: \"8. Publishing results of re-use\"Comment: What to do if there is discrepancy in findings between original and subsequent findings? Could undermine trust. Probably needs rows about “dispute” resolution.Yes, also very important and difficult to solve. Replication is very important in science (https://www.nature.com/news/1-500-scientists-lift-the-lid-on-reproducibility-1.19970) and given that the replication of complex and expensive experiments such as trials is not very much feasible, replication of the analysis is fundamental. We cannot think of any formal structure, to ‘referee’ disputes, that would be applicable here – any dispute would need to be played out in the literature, and each is likely to have different characteristics. We have restructured section 9 to add a row about the need to monitor disputes, as well as other possible consequences. Section: Table 2 Text ref: \"2. Locator services. Locator service for data sharing resources\"Comment: Will this be a familiar term to readers? I’m not sure what it means.We have tried to reword section 2 to make the meaning clearer. Trivial/Minor Section: Table 1 Comment: Would be quickly for each actor to find the role if this column was broken into separate columns, one per actor type, with the ticks for whether it is relevant. Table 1 ordered according to actor is an interesting proposal but according to our approach (list all processes/sub-processes following the clinical workflow) it would mean to add another table. We would not prefer to do that to keep the paper as simple as possible. Section: Table 1 Text ref: \"7.2 Agree an ID generation scheme for data objects\"Comment: “Data objects” needs a clear definition before the table. Perhaps a Glossary with the Abbreviations?Yes, a glossary with some main terms (defined as used in this paper) was added at the bottom of table 1." } ] }, { "id": "31018", "date": "20 Mar 2018", "name": "Matthias Löbe", "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, Ohmann et. al. perform a detailed analysis of steps required to share patient microdata from clinical trials with the research community. They provide a process diagram describing the workflow of preparing, transferring and maintaining the data and metadata to an external repository. The main part of the work consists of a comprehensive list of all sub-processes, the involved actors and services or tools. They also elaborate on scope and depth of the services or tools and give examples.\nThe valuable contribution of this work lies in the sequential structuring of data sharing tasks. Especially study groups who want (or have to) actively provide data have a checklist at hand, which gives them the opportunity to assess each sub-task in its complexity and to put together suitable persons or teams for implementation. This prevents important stakeholders from being overlooked or partial steps from being insufficiently taken into account, particularly with regard to regulatory issues.\nThe article focuses on aspects of data sharing in clinical trials, addressing a relevant problem of academic research, namely the long-term availability of research results in an environment that has only a limited lifespan due to project funding. It shows the complexity of the topic and every research group should already think about it during the project planning phase. Additionally, it is also relevant for other types of research projects, such as clinical registries, epidemiological cohorts or studies in health care research, with minor modifications.\nI particularly liked the fact that aspects of providing analysis environments were also addressed, e.g. with special Docker containers that bring the evaluation algorithms to the data instead of releasing data.\nThe weak part of the paper is that even with a detailed listing of the sub-processes and the relevant tools, most researchers will find it difficult to design a concrete implementation strategy or to check whether the implementation meets the state of the art. Notes such as \"Provide sample documents\", \"Assess risk of re-identification\" or \"Select suitable metadata schemas for object discovery\" are simply too vague to be a real help. At this point, a knowledge base must be built up that provides researchers with concrete guidelines, implementation guidelines and example scenarios for successful projects.\nPoints to address:\nThe workflow in Figure 1 assumes that the data set is only imported once into an external repository. However, there are many scenarios in which data sets will have to be updated or extended, e.g. in long-running investigations where interim evaluations are already being carried out. Snapshots of shared data must be saved for verification purposes.\n\nSome years ago, there has been an EMA draft policy on publication and access to clinical-trial data[1]. I’m not sure about the current status but it would be interesting to include the effort in this paper.\n\nPage 6, section 2.3.2 “Include request for broad consent for data sharing in informed consent documents.” The term broad consent might require a more detailed definition, because in Germany consent is always contextual and without specific and the ethics committees are looking into this.\n\nMetadata (sections 2.5, 5.4, 7.1) should not be limited to semantics and discovery. Another important topic for metadata is provenance metadata (measurement conditions, data quality, algorithms for calculated data)\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": [ { "c_id": "3594", "date": "20 Apr 2018", "name": "Christian Ohmann", "role": "Author Response", "response": "Our answer in bold and italics. In this paper, Ohmann et. al. perform a detailed analysis of steps required to share patient microdata from clinical trials with the research community. They provide a process diagram describing the workflow of preparing, transferring and maintaining the data and metadata to an external repository. The main part of the work consists of a comprehensive list of all sub-processes, the involved actors and services or tools. They also elaborate on scope and depth of the services or tools and give examples.The valuable contribution of this work lies in the sequential structuring of data sharing tasks. Especially study groups who want (or have to) actively provide data have a checklist at hand, which gives them the opportunity to assess each sub-task in its complexity and to put together suitable persons or teams for implementation. This prevents important stakeholders from being overlooked or partial steps from being insufficiently taken into account, particularly with regard to regulatory issues.The article focuses on aspects of data sharing in clinical trials, addressing a relevant problem of academic research, namely the long-term availability of research results in an environment that has only a limited lifespan due to project funding. It shows the complexity of the topic and every research group should already think about it during the project planning phase. Additionally, it is also relevant for other types of research projects, such as clinical registries, epidemiological cohorts or studies in health care research, with minor modifications.I particularly liked the fact that aspects of providing analysis environments were also addressed, e.g. with special Docker containers that bring the evaluation algorithms to the data instead of releasing data.The weak part of the paper is that even with a detailed listing of the sub-processes and the relevant tools, most researchers will find it difficult to design a concrete implementation strategy or to check whether the implementation meets the state of the art. Notes such as \"Provide sample documents\", \"Assess risk of re-identification\" or \"Select suitable metadata schemas for object discovery\" are simply too vague to be a real help. At this point, a knowledge base must be built up that provides researchers with concrete guidelines, implementation guidelines and example scenarios for successful projects. The purpose of the study was better explained at the end of the introduction. It was the objective to identify all processes/sub-processes involved in data sharing and to provide a classification of tools/services needed to support the processes. It is ground structuring work and it was not intended to provide specific help for data sharing (e.g. guidelines, examples). In a later stage of the CORBEL project concrete and speciifc tools/services to support data sharing will be made availalbe.Points to address: The workflow in Figure 1 assumes that the data set is only imported once into an external repository. However, there are many scenarios in which data sets will have to be updated or extended, e.g. in long-running investigations where interim evaluations are already being carried out. Snapshots of shared data must be saved for verification purposes.This is a relevant point and was included in the figure under 3) : Preparation of data sharing (after data collected or data update. Some years ago, there has been an EMA draft policy on publication and access to clinical-trial data[1]. I’m not sure about the current status but it would be interesting to include the effort in this paper.The EMA policy 70 is effective since January 2015 and applies to new drugs approved by the EMA after that date, thus only on a subset of trials testing pharmacological interventions. Moreover, the policy is only dealing with clinical study reports, i.e. aggregate data. Currently, the EMA is discussing the possibility of sharing individual participant data (IPD) from clinical trials. One EMA expert was included in our consensus exercise and one author of the current paper (CO) was invited to attend an EMA-workshop on anonymisation, 30.11.-1.12.2017). This publication could be used as input to an update of the EMA data sharing policy. This comment is added to the discussion. Page 6, section 2.3.2 “Include request for broad consent for data sharing in informed consent documents.” The term broad consent might require a more detailed definition, because in Germany consent is always contextual and without specific and the ethics committees are looking into this.The concept of broad consent has been discussed in detail in the BMJ Open paper published by the group in 2017.and was not tackled in this manuscript. Metadata (sections 2.5, 5.4, 7.1) should not be limited to semantics and discovery. Another important topic for metadata is provenance metadata (measurement conditions, data quality, algorithms for calculated data)Yes, provenance data are very important and an essential part of the metadata. We have added provenance metadata in 4.2.2 and 4.2.4." } ] } ]	1	https://f1000research.com/articles/7-138
https://f1000research.com/articles/7-200/v1	16 Feb 18	{ "type": "Research Note", "title": "A biochemical logarithmic sensor with broad dynamic range", "authors": [ "Steven A. Frank" ], "abstract": "Sensory perception often scales logarithmically with the input level. Similarly, the output response of biochemical systems sometimes scales logarithmically with the input signal that drives the system. How biochemical systems achieve logarithmic sensing remains an open puzzle. This article shows how a biochemical logarithmic sensor can be constructed from the most basic principles of chemical reactions. Assuming that reactions follow the classic Michaelis-Menton kinetics of mass action or the more generalized and commonly observed Hill equation response, the summed output of several simple reactions with different sensitivities to the input will often give an aggregate output response that logarithmically transforms the input. The logarithmic response is robust to stochastic fluctuations in parameter values. This model emphasizes the simplicity and robustness by which aggregate chemical circuits composed of sloppy components can achieve precise response characteristics. Both natural and synthetic designs gain from the power of this aggregate approach.", "keywords": [ "Biochemical circuit", "Hill equation", "synthetic biology", "systems biology", "aggregation", "robustness" ], "content": "Introduction\n\nI present a simple biochemical circuit that logarithmically transforms input signals. This circuit adds the outputs of several reactions that follow standard mass action Michaelis-Menton kinetics. Alternatively, the biochemical kinetics may follow the commonly observed Hill equation response, which includes Michaelis-Menton kinetics as a special case. This sensor has high dynamic range, responding logarithmically across many orders of magnitude. The high dynamic range is achieved by adding together reactions with different sensitivity ranges. The aggregate nature of this circuit provides robustness to parameter variations. Aggregate sensor design may explain the commonly observed high dynamic range of logarithmic biological responses and may also provide a useful tool for synthetic biology.\n\n\nResults and discussion\n\nMany biochemical reactions and cellular responses transform an input, x, into an output, y, according to the Hill equation\n\nThe output of the Hill equation scales approximately logarithmically with its input through the middle part of its response range, because y is roughly linear with respect to log x. Prior studies have emphasized that a Hill equation response can act as a logarithmic sensor3,4. However, a single Hill equation response provides a logarithmic sensor with limited dynamic range (Figure 1A).\n\nMy extended dynamic range sensor arises by adding together n Hill equations with increasing values of the half-maximal response, ci, as\n\nEach unit on the input scale corresponds to a doubling of the input. The range of 13 doublings is approximately four orders of magnitude, 213 ≈ 104. A logarithmic sensor responds linearly with respect to the logarithm of the input. The solid blue lines show the response, y, from Equation 1. The dashed gold lines are linear fits to the response. (A) A single Hill equation with c = 1 and k = 2. A linear response with low sensitivity occurs over a few doublings at small input levels. The following plots all use n = 7 and additional parameters as described from the summed Hill equation in Equation 1, in which ci = bi. (B) Response with k = 1 and b = 4. (C) Response with k = 2 and b = 4. (D) Response with k = 1 and b = 8. (E) Response with k = 2 and b = 8. (F) Same average parameters values of k = 2 and ci = bi for b = 4 as in the plot above, but with random parameter fluctuations around those average values. For each of the 7 Hill equations in the sum given in Equation 1, each parameter was obtained by a different random number drawn from a normal distribution. For each k, the parameters were drawn from a normal distribution with mean 2 and standard deviation 2 × 0.25 = 0.5. For each ci, the parameters were drawn from a normal distribution with mean 4i and standard deviation 4i ×0.25. The response remains nearly linear in spite of the random parameter fluctuations.\n\nFor example, if ci = bi, then each reaction in the sum has an increasing input value at which its maximal sensitivity occurs. Figure 1B–E shows that simple combinations of k and b create a logarithmic sensor, in which the output is linearly related to the logarithm of the input. The logarithmic relation holds robustly when the parameters k and b of the individual reactions vary stochastically (Figure 1F).\n\nSeveral biochemical circuit responses and many aspects of perception scale logarithmically5. A robust and generic pattern of this sort seems likely to depend on a robust and generic underlying design. In the search for a generic circuit, my biochemical logarithmic sensor has three advantages over prior designs. First, prior models depended on particular molecular assumptions about biochemical kinetics or reaction pathways3,4. My design requires only Michaelis-Menton or Hill equation responses, which are very widely observed in biochemical and cellular systems1. Second, prior models focused on single input-output processes, which have relatively narrow dynamic range. My aggregate design provides a logarithmic response over a vastly greater range. Third, the prior models’ responses are easily perturbed by parameter fluctuations. My design performs robustly with respect to broad fluctuations in parameters. The robustness of my logarithmic sensor emphasizes the potential to achieve precise response characteristics from underlying sloppy components when using an aggregate design6, 7.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nNational Science Foundation grant DEB–1251035 supports my research.\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\nZhang Q, Bhattacharya S, Andersen ME: Ultrasensitive response motifs: basic amplifiers in molecular signalling networks. Open Biol. 2013; 3(4): 130031. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrank SA: Input-output relations in biological systems: measurement, information and the Hill equation. Biol Direct. 2013; 8: 31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaniel R, Rubens JR, Sarpeshkar R, et al.: Synthetic analog computation in living cells. Nature. 2013; 497(7451): 619–623. PubMed Abstract \| Publisher Full Text\n\nOlsman N, Goentoro L: Allosteric proteins as logarithmic sensors. Proc Natl Acad Sci U S A. 2016; 113(30): E4423–E4430. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdler M, Alon U: Fold-change detection in biological systems. Curr Opin Syst Biol. 2018; 8: 81–89. Publisher Full Text\n\nFrank SA: The common patterns of nature. J Evol Bio. 2009; 22(8): 1563–1585. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrank SA: Evolution of robustness and cellular stochasticity of gene expression. PLoS Biol. 2013; 11(6): e1001578. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "30929", "date": "19 Mar 2018", "name": "Sudin Bhattacharya", "expertise": [ "Reviewer Expertise Computational biology and toxicology" ], "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 presents an interesting model of a robust biochemical logarithmic switch with a broad dynamical range as a sum of multiple Hill-equation based models.\nMy main problem with this work is the lack of a biological context with a real example (the author argues the model applies to natural as well as artificial circuits). Is the author proposing that the successive Hill-type switches would be arranged in series? A more natural example would be a layered cascade like a MAP kinase cascade1. But in that case the output would not be a simple linear sum as in the author’s Eqn. (1), but rather something like a Hill function of a Hill function.\n\nThe contention that “My design performs (more) robustly with respect to broad fluctuations in parameters” should be established more rigorously. How were the stochastic simulations performed? It’s hard to tell since no methodological details are provided.\n\n“Menten” in Michaelis-Menten mis-spelt throughout manuscript (after Maud Menten: https://en.wikipedia.org/wiki/Maud_Menten)\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? No\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? No", "responses": [ { "c_id": "3523", "date": "23 Mar 2018", "name": "Steven Frank", "role": "Author Response F1000Research Advisory Board Member", "response": "I thank Dr. Bhattacharya for sharing his expertise on topics related to the important Hill equation (see ref. 1 of the article). Dr. Bhattacharya made three helpful comments in his review, which I respond to in reverse order. First, I appreciate the spelling correction. I had reread the interesting history of the Michaelis-Menten equation when preparing this article, but nonetheless incorrectly spelled Menten's name. I have fixed that misspelling in the revision. Second, I have uploaded the source code, as a supplementary file, that I used to do the stochastic simulations. The short, simple code provides a clear, exact description of the methods. The code was used to generate Figure 1, and can be used to regenerate that figure and explore alternative assumptions. A summary of the methods are provided in the legend for Figure 1. The final point concerns the lack of specific biological examples and details. The first aspect of this comment is a question asked by Dr. Bhattacharya: \"Is the author proposing that the successive Hill-type switches would be arranged in series?\" I am proposing a summation of individual responses to a stimulus, as described in Equation 1. For example, suppose that a set of sensors respond to sound intensity. Each sensor has a Hill-like response curve, but tuned to different intensity level. The output of the aggregate set of sensors is the sum of the individual sensor responses. Each sensor may comprise a simple or complex set of reactions. All that we care about here is that the net response per sensor is Hill-like in shape. That same logic could describe the response to a molecular signal. Various reactions could each have a Hill equation response, but tuned to different sensitivities. The aggregate consequence of those responses would be a net output level. With regard to an explicit example, I gave a lot of thought to that issue while preparing the manuscript. I could not find a compelling example for which sufficient biological detail about mechanism had been described. There are plausible cases that are worth exploring, such as the sensory problems that I mentioned. For molecular systems, there are hormones for which cells have multiple receptors, presumably tuned differently with regard to response and other aspects (e.g., estrogen receptors). However, I agree that those multireceptor systems do not provide a compelling case given current knowledge. Why are there no compelling molecular examples? I think the reason is that researchers rarely look for responses that arise by aggregate processes, such as summation of underlying reactions. Instead, the search is almost always for a single reaction or explicit pathway that exactly matches the desired input-output pattern. So my goal for this article is to suggest a plausible alternative for consideration. One advantage of publishing with F1000Research is that I can update the article in the future. If a good example arises in discussion with readers of the article or from the literature, I will update the article to include that example. For now, I think it is most important that the novel idea be published in a concise and direct manner, with emphasis on the simple logic of the model." } ] } ]	1	https://f1000research.com/articles/7-200
https://f1000research.com/articles/7-477/v1	18 Apr 18	{ "type": "Research Article", "title": "Switching from biosimilar (Basalin) to originator (Lantus) insulin glargine is effective in Chinese patients with diabetes mellitus: a retrospective chart review", "authors": [ "Xia Hu", "Lei Zhang", "Yanhu Dong", "Chao Dong", "Jikang Jiang", "Weiguo Gao", "Xia Hu", "Lei Zhang", "Yanhu Dong", "Chao Dong", "Jikang Jiang" ], "abstract": "Background: This study investigated the effectiveness and safety of switching from Basalin® to Lantus® in Chinese patients with diabetes mellitus (DM). Methods: A retrospective chart review conducted using the electronic medical records of patients hospitalized at the Qingdao Endocrine and Diabetes Hospital from 2005 to 2016. All patients were diagnosed with DM and underwent switching of insulin from Basalin to Lantus during hospitalization. Data collected included fasting (FBG), pre- and post-prandial whole blood glucose, insulin dose, reasons for insulin switching and hypoglycemia. Four study time points were defined as: hospital admission, Basalin initiation, insulin switching (date of final dose of Basalin), and hospital discharge. Blood glucose measurements were imputed as the values recorded closest to the dates of these four time points for each patient. Results: Data from 73 patients (70 patients with type 2 diabetes, 2 with type 1, and 1 undisclosed) were analyzed. At admission, mean glycated hemoglobin (HbA1c) and FBG were 8.9% (SD=1.75) and 9.98 (3.22) mmol/L, respectively. Between Basalin initiation and insulin switch, mean FBG decreased from 9.68 mmol/L to 8.03 mmol/L (p<0.0001), over a mean 10.8 (SD=6.85) days of Basalin treatment, and reduced further to 7.30 mmol/L at discharge (p=0.0116) following a mean 6.6 (7.36) days of Lantus. The final doses of Basalin and Lantus were similar (0.23 vs. 0.24 IU/kg/day; p=0.2409). Furthermore, reductions in pre- and post-prandial blood glucose were also observed between Basalin initiation, insulin switch and hospital discharge. The incidence of confirmed hypoglycemia was low during Basalin (2 [2.4%]) and Lantus (1 [1.2%]) treatment, with no cases of severe hypoglycemia. Conclusion: In this study population, switching from Basalin to Lantus was associated with further reductions in blood glucose, although the dose of insulin glargine did not increase. Further studies are required to verify these findings and determine the reason for this phenomenon.", "keywords": [ "Insulin glargine", "biosimilar", "diabetes mellitus", "Lantus", "Basalin" ], "content": "Introduction\n\nIn China, insulin is more widely used to treat patients with type 2 diabetes mellitus (T2DM) compared with many Western countries1,2. Chinese patients with T2DM are characterized by a relatively young age of diabetes onset, low bodyweight, and early β-cell dysfunction combined with insulin resistance2. Furthermore, progressive deterioration of β-cell function is known to contribute to the increasing difficulty of achieving glycemic control experienced during the T2DM disease course3–5. Therefore, interventions to preserve β-cells are recognized to be effective and, considering the early β-cell dysfunction which characterizes Chinese T2DM patients, can be especially effective in this population6. However, challenges for implementing insulin treatment in China include lack of patient education, limitations of medical resources, inadequate health care systems making patient follow up difficult, and suboptimal blood glucose monitoring1. Therefore, Chinese physicians often prefer to initiate intensified treatment regimens in an inpatient setting to allow close control of treatment, and monitoring of effectiveness and safety. Given this situation, early initiation of insulin treatment in an inpatient setting is often favored in China for the treatment of patients with T2DM. Several studies in Chinese patients with newly-diagnosed T2DM have shown that early intensive insulin treatment preserves β-cell function and leads to glycemic remission7–9. Furthermore, a meta-analysis of previous studies showed that 46.3% of Chinese patients with T2DM who received intensive insulin treatment achieved glycemic remission after 12 months, without the use of anti-diabetic medication and relying solely on lifestyle modification to control blood glucose levels10.\n\nBasal insulin is recommended as a convenient way to initiate insulin treatment by most international treatment guidelines, and by the Chinese Diabetes Association11–13. (see the International Diabetes Federations Diabetes Atlas, 7th edition) As reported by the large, observational ORBIT study, insulin glargine is the most popular basal insulin for the initiation of insulin treatment in Chinese patients with T2DM (71%), followed by detemir (13%) and neutral protamine Hagedorn (NPH)(16%) insulin. In China, there are two available versions of insulin glargine; the originator Lantus® (Sanofi), and the biosimilar Basalin® (see Gan & Lee Pharmaceuticals history page), which has been commercially available since 2005 in China, Egypt, Pakistan, South East Asia, and countries in Latin America14. Basalin was the first biosimilar of insulin glargine marketed in China, and has since become a commonly used treatment option for many Chinese physicians. However, while biosimilars are developed to be highly similar to originator therapies, due to the degree of natural variability inherent to biological drugs, which are produced using living organisms, and because of differences in manufacturing processes between products, they are not identical to the original therapy15. Results from a previous head-to-head study of Basalin and Lantus in Chinese people with T2DM showed equivalent glycemic control and safety, and a further crossover study in Chinese people with T2DM using a continuous glucose monitoring system (CGMS) reported non-inferiority of Basalin to Lantus for mean blood glucose, blood glucose fluctuations and rates of hypoglycemia16,17. However, other than these two previous reports, there are no further confirmatory studies in real-world clinical practice. In addition, a local-language case report described improved blood glucose control and reduced insulin dose in three Chinese patients with T2DM who switched from Basalin to Lantus due to suboptimal glycemic control18. Given this situation, it is important to further verify the findings of this case report in a larger population of patients who switched from Basalin to Lantus in a real-life clinical practice setting. We therefore hypothesized that patients with diabetes who switch from Basalin to Lantus in a real-world setting may achieve improved control of blood glucose with a similar safety profile.\n\n\nMethods\n\nThis was a retrospective chart review conducted based on the electronic medical records (EMRs) of patients hospitalized at the Qingdao Endocrine and Diabetes Hospital from 2005 to 2016. Qingdao Endocrine and Diabetes Hospital is a public, non-profit, tertiary hospital that was established in 2002 and is located in Qingdao city, one of the biggest cities on the east coast of China. The Hospital is the only specialist diabetes hospital, not only in Qingdao, but also throughout the region, with a catchment area of approximately 100 kilometers, which covers 7.2 million people. The majority of the patients treated in the Hospital are Han Chinese residing in Qingdao city and the surrounding regions. Less than 1% of patients are national ethnic minorities or of foreign origin. In general, patients are hospitalized due to suspected acute diabetic complications including ketosis, ketoacidosis, diabetic hyperosmolality, uncontrolled hyperglycemia and at least one chronic complication, or significant worsening of chronic complications of diabetes (including rental hemorrhage, significant elevated albuminuria and/or serum creatinine, acute myocardial infarction, etc.).\n\nThe data selection process is summarized in Figure 1. EMRs were first searched using the keywords “Lantus”, “Basalin” or “insulin glargine” to identify EMRs with recorded use of insulin glargine. EMRs were then subdivided based on the type of insulin glargine used; Lantus and Basalin (103), Basalin only (690), Lantus only (4311), or no information about which type of insulin glargine had been used (3). The EMRs with recorded use of both Lantus and Basalin were further sorted into two groups; switch from Basalin to Lantus (82) and switch from Lantus to Basalin (21). Finally, the EMRs were evaluated by hand to exclude cases involving multiple switches between Basalin and Lantus, and identify multiple EMRs for the same patient on different hospitalizations (in such instances only the case from the most recent hospitalization was included in the analysis). Of the 21 EMRs including a switch from Lantus to Basalin, nine reported multiple switches of insulin, and therefore only 12 EMRs (each representing one patient) were eligible for inclusion, and these data were not included in the present analysis. Of the 82 cases including switching from Basalin to Lantus, six records included multiple switches of insulin, and one subject had been hospitalized four times, and therefore had four medical records; therefore, a total of 73 patients who switched from Basalin to Lantus were included.\n\nThe key objective of the present analysis was to evaluate the change in FBG for patients with DM after switching from Basalin to Lantus. Secondary aims of the analysis included evaluation of changes in seven-point blood glucose, basal and prandial insulin dose, and hypoglycemia after switching from Basalin to Lantus. Four study time points were defined as hospital admission, Basalin initiation, insulin switching (the date of the final dose of Basalin), and hospital discharge (Figure 2). For each study subject, values of blood glucose at hospital admission and hospital discharge were imputed as the measurements recorded closest to the date of hospital admission and discharge, and values at Basalin initiation and insulin switch were defined as the measurements recorded closest to the date of the first and last doses of Basalin, respectively. The duration of insulin glargine treatment was calculated as the end date of the final dose minus the start date of the initial dose +1 day. If the date of the final dose of Lantus was not recorded then the final dose date was taken as the date of hospital discharge.\n\nThe study protocol was approved by the Ethical Review Board of Qingdao Endocrine and Diabetes Hospital in a regular meeting during October 2016 (Document No 2016-10-1). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964, as revised in 2013. As this was a retrospective observational study based on electronic medical records it was not possible to contact each patient for individual consent, however the researchers received anonymized data from the hospital's IT department with permission from both the Ethical Review Board and the Principal of the Hospital.\n\nBasalin (Gan & Lee Pharmaceutical, Beijing, China) and Lantus (Sanofi, Paris, France) are both provided in a 100 IU/mL concentration by the manufacturers. Both insulin glargines are provided with an injection pen; the GanLee Pen (Gan & Lee Pharmaceutical) for Basalin and the SoloSTAR®/ClikSTAR® (Sanofi) for Lantus.\n\nData were retrospectively harvested from EMRs collected in the hospital information system and included demographics, medical history, details of medication including pre-hospitalization treatment regimen and concomitant use of oral antidiabetic drugs (OAD), laboratory measurements such as glycated hemoglobin (HbA1c) and daily blood glucose measurements, and safety data. Raw data were encoded by a trained member of staff. Daily blood glucose control was evaluated using seven-point blood glucose, comprised of fasting blood glucose levels (FBG) before breakfast, and blood glucose levels post-breakfast, pre- and post- lunch and dinner, and pre-bed.\n\nBlood glucose measurements were conducted by hospital staff from finger-stick blood samples and using the Nova StatStrip system (Nova Biomedical, MA, USA). Hypoglycemia was defined as any recorded incidence of blood glucose ≤3.9 mmol/L, and severe hypoglycemia as blood glucose ≤2.8 mmol/L.\n\nTwo subgroup analyses were performed; in patients who received basal-bolus therapy with Basalin and prandial insulin as their initial therapy (excluding patients who received OADs), and for patients who switched from Basalin to Lantus due to suboptimal glycemic control as recorded in their medical record.\n\nData were summarized using descriptive statistics. Continuous variables are presented as mean (standard deviation [SD]), and discrete variables are summarized as frequency and percentage. Paired Student’s t-tests were used to evaluate differences between variables. A p-value <0.05 was considered statistically significant. All analyses were conducted using SAS software (version 9.4).\n\n\nResults\n\nAt baseline, the majority of the study patients were middle aged or older, with a mean body mass index of 27.4 kg/m2 and a mean duration of diabetes mellitus (DM) of 9.1 years (Table 1). Metabolic control was poor in most patients, indicated by an average HbA1c of 8.9%, and FBG of 9.98 mmol/L, at hospital admission. Diabetic complications had been reported in 86.3% of patients, and 38.4% had a history of cardiovascular disease. The mean duration of hospitalization was 22.3 days. Initial treatment with Basalin insulin glargine was received in combination with prandial insulin for the majority of patients (84.9%). The most common reason for switching to Lantus as recorded in patients’ medical records was suboptimal glycemic control (30.1%), followed by patient decision (4.1%).\n\naData are summarized as mean (SD) unless stated; bAny diabetic complication, yes or no;\n\ncpatients may have received other treatment before initiating biosimilar insulin glargine.\n\nOADs, oral antidiabetic drugs.\n\nOverall, mean FBG decreased from initiation of Basalin insulin glargine until hospital discharge (Table 2). From initiation of treatment with Basalin insulin glargine until insulin switch, FBG decreased by 1.65 mmol/L; from 9.68 mmol/L to 8.03 mmol/L (p<0.0001). From insulin switch until hospital discharge (while patients were receiving Lantus), FBG reduced by a further 0.73 mmol/L, to 7.30 mmol/L (p=0.0116).\n\naData are summarized as mean (SD) unless stated; bcalculated using a two-tailed Student’s t-test.\n\nBG, blood glucose; FBG, fasting blood glucose.\n\nMean post-prandial blood glucose values indicated improved glycemic control during treatment with Basalin, and further improvements after switching to Lantus (Table 2, Supplementary Figure 1). Seven-point blood glucose decreased at all four study time points from hospital admission to hospital discharge. Between Basalin initiation and insulin switch the reductions in mean seven-point blood glucose were significant across all seven blood glucose measurements (p<0.05). Subsequently, between insulin switch and hospital discharge, reductions in mean seven-point blood glucose values were significant (p<0.05) at all times except post-lunch and pre-dinner.\n\nSubjects received Basalin insulin glargine for a mean duration of 10.8 (6.85) days, and during this time the mean insulin dose increased significantly from 0.19 IU/kg/day to 0.23 IU/kg/day (p<0.0001)(Table 3). Lantus insulin glargine was received for a mean duration of 6.6 (7.36) days; the initial mean dose was 0.24 IU/kg/day and the final dose before hospital discharge was 0.24 IU/kg/day (p=0.8720). In addition, the mean final doses of Basalin and Lantus were similar (p=0.2409).\n\naPaired Student’s t-test; bfor patients receiving basal + prandial regimens.\n\nFor patients receiving prandial insulin, the mean dose of prandial insulin increased from 0.32 IU/kg/day to 0.40 IU/kg/day between the initial and final dose of Basalin (p<0.0001), and decreased from 0.41 IU/kg/day to 0.40 IU/kg/day between initial and final dose of Lantus (p<0.4830).\n\nThe 62 patients who received basal-bolus (basal insulin plus multiple daily injections of prandial insulin) therapy experienced significant reductions in FBG from initiation of Basalin to hospital discharge. Between initiation of Basalin therapy and insulin switch mean FBG decreased from 10.03 mmol/L to 8.25 mmol/L (p=0.0001), and from insulin switch to hospital discharge decreased further to 7.49 mmol/L (p=0.0217) (Table 4, Supplementary Figure 2). In this subgroup, overall glycemic control was also improved during treatment with Basalin, and continued to improve after switching to Lantus. Between initiation of Basalin and insulin switch, seven-point blood glucose reduced significantly at all seven measurements, and from insulin switch to hospital discharge decreased significantly at all measurements except post-lunch and pre- and post-dinner. The final mean doses of Basalin and Lantus were similar (0.24 [0.08] IU/kg/day vs. 0.25 [0.09] IU/kg/day; p=0.1321), and the final mean dose of prandial insulin at the last administrations of Basalin (0.4 [0.15]) and Lantus (0.4 [0.16]) were also similar (p=0.8429) (Supplementary Table 1).\n\nAmong the 22 patients who switched to Lantus due to suboptimal glycemic control as recorded in their medical records, FBG decreased from 10.17 (3.59) mmol/L to 8.51 (2.18) mmol/L (p=0.0540) during treatment with Basalin, and between insulin switch and hospital discharge decreased further to 7.37 (1.70) mmol/L (p=0.0337) (Supplementary Table 2). Among these patients, an overall trend towards reduction in seven-point blood glucose was also observed. Similar to the results in the main analysis population, the mean final doses of Basalin and Lantus were comparable (0.25 [0.08] IU/kg/day vs. 0.26[0.1] IU/kg/day; p=0.5069), as were the mean final doses of prandial insulin at final administrations of Basalin and Lantus (0.43 (0.13) IU/kg/day vs. 0.41 [0.14] IU/kg/day; p=0.5085) (Supplementary Table 3).\n\nHypoglycemia was reported by 2 (2.4%) of patients during treatment with Basalin insulin glargine, and by 1 (1.2%) of patients during treatment with Lantus. No patients experienced severe hypoglycemia during hospitalization.\n\n\nDiscussion\n\nOur retrospective study found that Chinese patients with T2DM who initiated Basalin with pre-prandial insulin or OADs achieved further reductions in overall hyperglycemia, especially fasting hyperglycemia, after switching to Lantus (8.03 mmol/L vs. 7.30 mmol/L; p=0.0116) with a similar mean basal insulin dose (0.23 IU/kg/day vs. 0.24 IU/kg/day; p=0.8720). This result is consistent with a previous case series that found three Chinese patients with T2DM inadequately controlled with Basalin achieved improvements in glycemic control after switching to Lantus, with a similar dosage of insulin18,19. Patients in our study were relatively young (59.8 years) with a moderate disease course (9.1 years), poor glycemic control (HbA1c of 8.9%) and a high proportion of existing diabetic complications (86.3%) but a comparatively low prevalence of cardiovascular disease (38.4%). This may explain why the clinical decision was made to switch to Lantus, after achieving sub-optimal glycemic control with Basalin.\n\nIn contrast to our findings, a recently published blinded crossover study showed non-inferiority in terms of mean blood glucose, glucose fluctuations and rates of hypoglycemia as measured by CGMS for Chinese outpatients with T2DM receiving Basalin or Lantus16. Although patients in this previous study had a similar mean age, BMI and duration of T2DM as our study, there were several important differences. Firstly, patients in this previous study had a relatively high mean blood glucose level during the study period in both the Basalin group and Lantus group (9.35 mmol/L vs. 9.67 mmol/L, respectively; p=0.387), especially a high FBG concentration of nearly 10 mmol/L. Secondly, no insulin titration scheme was used and it was not a treat-to-target study, in contrast to our study which utilized insulin dose titration. Finally, the duration of the previous study was 5 days with crossover on day 3, and 2 days of insulin administration before and after cross over, which may not have allowed adequate time for a full washout of the initial insulin type.\n\nPatients in this study had poor glycemic control at Baseline (HbA1c=8.9%) and required intensified therapy to rapidly lower blood glucose levels, despite the majority having already received treatment for T2DM. Previous studies in hospitalized Chinese patients with poorly-controlled T2DM receiving intensive insulin therapy have shown that glycemic control targets based on blood glucose levels (FBG and post-prandial glucose [PPG]) can be reached in around 4 days20,21. In comparison, the mean duration of Basalin (10.8 days) and Lantus (6.6 days) insulin glargine in the present study was sufficient to allow glycemic control targets to be reached. Additionally, it should be noted that given the short-term nature of the intensified insulin treatment being assessed in this study, the use of FBG as the primary assessment of glycemic control is justified versus other longer-term biomarkers such as HbA1c, and a precedent has been set for this in previous studies of short-term intensive insulin treatment7–9.\n\nEvidence suggests that intensive insulin treatment can protect and improve β-cell function through achievement of optimal glycemic control10,22,23. Furthermore, initiation of a basal-bolus insulin regimen is recommended by most treatment guidelines for treatment intensification in patients with T2DM who cannot achieve glycemic control with OADs and basal insulin and is also recommended for hospitalized patients11–13,24. The majority of patients in this study initiated treatment with a basal-bolus insulin regimen and achieved reductions in mean seven-point blood glucose during Basalin treatment, and further reductions following the switch to Lantus with a similar final dose of prandial insulin to that recorded at the end of Basalin treatment (0.40 IU/kg/day at both time points). This suggests that the further reductions in FBG achieved with Lantus treatment contributed to the overall control of hyperglycemia. As the glycemic profile of diabetes patients consists of basal glycemia, basal hyperglycemia and postprandial hyperglycemia, reductions of “basal” hyperglycemia which is often expressed as “fasting” blood glucose levels have been hypothesized to contribute to a reduction of post-prandial hyperglycemic excursions25. In addition, despite the use of an intensive basal-bolus regimen in the majority of patients, the incidence of confirmed hypoglycemia was similarly low during treatment with Basalin and Lantus (2.4% vs. 1.2%).\n\nTo determine the potential confounding effects of concomitant OAD and prandial insulin use on glycemic control associated with basal insulin , we conducted two subgroup analyses in patients treated with a purely basal-bolus regimen with no OAD use and patients with recorded switching from Basalin to Lantus due to suboptimal glycemic control, and in both of these subgroups mean FBG was lower after switching from Basalin; 8.25 mmol/L vs. 7.49 mmol/L (p=0.0217) and 8.51 mmol/Lvs.7.37 mmol/L (p=0.0337). Furthermore, the results of the Joint Asia Diabetes Evaluation (JADE) study also reported that biosimilar insulin use in Asia is associated with suboptimal glycemic control, indicated by higher FBG and HbA1c levels and a lower proportion of patients achieving glycemic control targets (HbA1c <7.0%) compared with originator insulin (13.6% vs. 17.2%)19.\n\nThere are several limitations of this analysis which deserve discussion. Firstly, this was a retrospective study that had many potential confounding factors including patient age, duration of DM, diabetic complications and co-morbidities, which may have affected treatment outcomes, insulin dose and safety. However, patients acted as their own controls, which reduces the effect of these confounding factors somewhat, particularly because most of the confounding factors would be constant over the relatively short mean durations of treatment and hospital stay. Secondly, all patients in this study were hospitalized with strictly-controlled diet, exercise and monitoring of blood glucose, which would be expected to have a positive effect on glycemic control independently of basal insulin treatment, and it may also be hypothesized that switching to a different basal insulin perceived as ‘superior’ could lead to a placebo effect. Although these are potential confounding factors for attributing improvements in glycemic control to basal insulin treatment alone, it should be noted that patients achieved sub-optimal glycemic control with Basalin during the initial part of their hospital stay (an average of 10 days) during which modified diet and lifestyle would be expected to have the largest positive impact on blood glucose levels. Finally, although this was a single-center study with limited data the patient profile is similar to that reported in the Fine Asia and ORBIT studies, which indicated our findings potentially have treatment implications generalizable to the broader population of Chinese patients with T2DM26,27.\n\nIn conclusion, this study provides evidence from real-world clinical practice that switching from Basalin to Lantus is associated with an improvement in blood glucose levels, with a similar insulin dose, in hospitalized patients with T2DM. Despite the limitations of the present analysis, these findings warrant further investigation by crossover randomized controlled study or further real-world evidence from a larger population.\n\n\nData availability\n\nDataset 1: Switching from biosimilar (Basalin) to originator (Lantus) insulin glargine, propensity score matching with readme file explain the analysis 10.5256/f1000research.13923.d19967428", "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 – Document containing the following Tables and Figures:\n\nClick here to access the data.\n\nSupplementary Table 1. Summary of insulin doses during initial treatment with Basalin insulin glargine and after switching to Lantus for patients who received basal-bolus therapy only without OADs\n\nSupplementary Table 2. Seven-point blood glucose summary for patients who switched due to suboptimal glycemic control as recorded in medical records\n\nSupplementary Table 3. Summary of insulin doses during initial treatment with Basalin insulin glargine and after switching to Lantus for patients who switched to Lantus due to suboptimal glycemic control as recorded in medical records\n\nSupplementary Figure 1. Seven-point blood glucose measurement for Chinese patients with diabetes mellitus at hospital admission, Basalin insulin glargine initiation, insulin switch, and hospital discharge\n\nSupplementary Figure 2. Seven-point blood glucose measurement for Chinese patients with diabetes mellitus who received basal-bolus treatment only with no OAD use (n=62) at hospital admission, Basalin insulin glargine initiation, insulin switch, and hospital discharge\n\n\nReferences\n\nJi L, Newman J, Lu J, et al.: Understanding the standard of care in the treatment of type 2 diabetes in China: results from a national survey. Chin Med J (Engl). 2014; 127(20): 3524–9. PubMed Abstract\n\nMa RC, Chan JC: Type 2 diabetes in East Asians: similarities and differences with populations in Europe and the United States. Ann N Y Acad Sci. 2013; 1281: 64–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTurner RC, Cull CA, Frighi V, et al.: Glycemic control with diet, sulfonylurea, metformin, or insulin in patients with type 2 diabetes mellitus: progressive requirement for multiple therapies (UKPDS 49). UK Prospective Diabetes Study (UKPDS) Group. JAMA. 1999; 281(21): 2005–12. 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PubMed Abstract \| Publisher Full Text\n\nZhang T, Lin M, Li W, et al.: Comparison of the Efficacy and Safety of Insulin Detemir and Insulin Glargine in Hospitalized Patients with Type 2 Diabetes: A Randomized Crossover Trial. Adv Ther. 2016; 33(2): 178–85. PubMed Abstract \| Publisher Full Text\n\nChoi SB, Lee JH, Lee JH, et al.: Improvement of β-cell function after achievement of optimal glycaemic control via long-term continuous subcutaneous insulin infusion therapy in non-newly diagnosed type 2 diabetic patients with suboptimal glycaemic control. Diabetes Metab Res Rev. 2013; 29(6): 473–82. PubMed Abstract \| Publisher Full Text\n\nRetnakaran R, Drucker DJ: Intensive insulin therapy in newly diagnosed type 2 diabetes. Lancet. 2008; 371(9626): 1725–6. PubMed Abstract \| Publisher Full Text\n\nAmerican Diabetes Association: 6. Glycemic Targets. In Standards of Medical Care in Diabetes-2017. Diabetes Care. 2017; 40(Suppl 1): S48–S56. PubMed Abstract \| Publisher Full Text\n\nMonnier L, Colette C, Owens D: Postprandial and basal glucose in type 2 diabetes: assessment and respective impacts. Diabetes Technol Ther. 2011; 13 Suppl 1: S25–32. PubMed Abstract \| Publisher Full Text\n\nJi L, Zhang P, Zhu D, et al.: Observational Registry of Basal Insulin Treatment (ORBIT) in patients with type 2 diabetes uncontrolled with oral antihyperglycaemic drugs: Real-life use of basal insulin in China. Diabetes Obes Metab. 2017; 19(6): 822–830. PubMed Abstract \| Publisher Full Text\n\nTsai ST, Pathan F, Ji L, et al.: First insulinization with basal insulin in patients with Type 2 diabetes in a real-world setting in Asia. J Diabetes. 2011; 3(3): 208–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu X, Zhang L, Dong Y, et al.: Dataset 1 in: Switching from biosimilar (Basalin) to originator (Lantus) insulin glargine is effective in Chinese patients with diabetes mellitus: a retrospective chart review. F1000Research. 2018. Data Source" }	[ { "id": "33295", "date": "03 May 2018", "name": "Longyi Zeng", "expertise": [ "Reviewer Expertise Diabetes" ], "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 is well structured with a clear objective to explore a very common scientific question in daily clinical practice. In this study, authors reported a significant FBG reduction(additional -0.67mmol/L) and better overall hyperglycemia control after switching from Basalin to Lantus at similar insulin dosage in hospitalized diabetes patients, mostly are type 2 diabetes patients. This outcome helps provide an initial answer on the efficacy and safety difference between biosimilar insulin and originator insulin in real clinical practice for physicians. This study also implies the importance of FBG control on overall glycemic control for physicians.\n\nAs a retrospective chart study, the outcomes inevitably limited by some confounding factors as discussed in the paper. To better verify the outcome, authors explored subgroup analysis in basal bolus regimen and recorded suboptimal glycemic control population, from which with similar outcomes as in total population. Sufficient and valid data in Lantus switching to Basalin as control group or crossover design study may provide further useful and robust information on efficacy, safety evaluation.\n\nAfter a careful review of this manuscript, I do think it appropriate for publication in F1000 Research.\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": "33298", "date": "04 May 2018", "name": "Benli Su", "expertise": [ "Reviewer Expertise Clinical endocrinology and diabetes" ], "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 peakless long-action insulin analogue, Insulin glargine has been widely used in China, as basal insulin initiating paradigm become more and more accepted. Biosimilar insulin glargine has been on market for many years. As debate if biosimilar is as effective as originator continued, we also once noticed three patients switching from biosimilar glargine to originator resulted in improved glycemic control without increase in dosage. In the report the authors retrieved data of inpatient electronic record, studied retrospectively if the switching from biosimilar insulin glargine to its originator would improve patients glycemic control. They found it did improve patient glucose control after the switching. The authors discussed the possible reasons. But as reported, this is a retrospectively study, there was no control. From the authors schematic description of data retrieval, we noticed there still had around 20 patients who switched their insulin from originator to biosimilar, although the number was small, this might still be used as a control. We discussed our case report one possible reason of improvement in glucose control might be due to inpatient diabetes education, strict dietary restriction. The adding of these “control” patients would provide more strong evidence supporting the use of originator.\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": [] }, { "id": "33296", "date": "08 May 2018", "name": "Yan Li", "expertise": [ "Reviewer Expertise Prevention and treatment of diabetes mellitus and complications", "thyroid diseases", "adrenal diseases" ], "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 explored 73 diabetes patients treated with basal insulin regimen, starting from Basalin and then switched to Lantus in hospitalized setting based on electronic medical records from 2005 to 2016. The main outcomes showed further fasting blood glucose reduction with similar insulin required and similar safety profile. Meanwhile, the study released the same message that basal insulin was not fully titrated as other studies in Chinese diabetes patient, only about 0.05U/kg insulin dose increased (0.19U/kg to 0.24U/kg) at discharge compared with prandial insulin (0.18U/kg insulin dose increased from 0.32 to 0.40 IU/kg) after almost three weeks of hospitalization. The titration situation was supposed to be related to the limited clinical experience of the two basal insulin, Lantus and Basalin, which was launched in 2004, 2005 respectively and data included for analysis between 2005 and 2016, with diversified practice. But the outcomes did reflect the effectiveness of Lantus and Basalin in real life.\n\nIn addition to the limitation mentioned in this article, some limitation should also be considered. First, the treatment duration of Lantus was 6.6(7.36),which meant the minimum treatment duration might be about 1-2 day. Therefore, it is much more appropriate to explore the data in steady state,for example 4-5 day after the first dosing. Second, the data was from a single site and the patient number was small for a retrospective study to address a strong conclusion but authors has realized it and provided a quite fair conclusion on the effectiveness.\n\nThe article was well written and I personally agree it can be considered for publication in your journal.\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": "33299", "date": "11 May 2018", "name": "Xia 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\nI recommend the indexing of this paper in your journal based on the following considerations:\n1.\n\nThis is one of the few papers discussing the effectiveness and safety between biosimilar insulin and originator insulin in real world clinical practice in a Chinese diabetes population. It releases the message that biosimilar is potentially different in effectiveness from originator as reported by previous papers. It helps provide a clinical implication to physicians and raise the necessity of long term observation or well designed trials to identify the differences in real world at the same time.\n\n2.\n\nDespite the limitation of small sample size, short term treatment duration and potential bias mentioned in this paper, the authors smartly designed two subgroup analysis. It also showed that the mean FBG was lower after switching from Basalin to Lantus , 8.25mM vs. 7.49mM(p=0.0217,for purely basal-bolus regimen with no OAD population) and 8.51mM vs.7.37mM(p=0.0337,for recorded suboptimal glycemic control population). The conclusion is consistent as that in the whole group.\n\n3.\n\nThe graphs should be displayed in a simple way to be well understood.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-477
https://f1000research.com/articles/7-475/v1	18 Apr 18	{ "type": "Case Report", "title": "Case Report: Haemolytic anaemia with ceftazidime use in a patient with cystic fibrosis", "authors": [ "Jun Yong", "Freddy Frost", "Dilip Nazareth", "Martin Walshaw", "Freddy Frost", "Martin Walshaw" ], "abstract": "Drug-induced Immune Haemolytic Anaemia (DIIHA) is a rare but serious complication of cephalosporin use. Ceftazidime is recognized to be a rare cause of DIIHA. We report and discuss a case of DIIHA in a person with cystic fibrosis who developed severe haemolytic anaemia following use of ceftazidime in the management of an acute pseudomonal pulmonary exacerbation.", "keywords": [ "Ceftazidime", "Cephalosporins", "Drug Induced Immune Haemolytic Anaemia" ], "content": "Introduction\n\nDrug-Induced Immune Haemolytic Anaemia (DIIHA) is a serious but uncommon complication, with an estimated incidence of 1 per million of the population1,2. Since their introduction in the early 1960s, cephalosporins have been well established causes of DIIHA, most commonly with the third generation cephalosporins, cefotetan and ceftriaxone.\n\nCeftazidime is often a first choice cephalosporin to treat pulmonary exacerbations due to Pseudomonas aeruginosa in people with cystic fibrosis (CF), but despite its frequent use DIIHA is rare, with only 4 (non-fatal) cases being reported between 1971 to 20142–4. For the first time, we present a case of DIIHA secondary to intravenous (IV) ceftazidime use in a person with CF.\n\n\nCase report\n\nA 23 year old female with CF (DF508/DF508) chronically infected with Pseudomonas aeruginosa presented with a 2 week history of general malaise, increasing shortness of breath, a productive cough and a decline in FEV1. She began in-patient treatment for a pulmonary exacerbation and was commenced on a 14 day course of two antipseudomonal IV antibiotics (3g of Ceftazidime and 2 Mega-Units of Colistimethate Sodium three times a day) in line with accepted CF practice. Following initial improvement, on day 4 she developed new pyrexia, hypotension, tachycardia and jaundice, and had an increasing oxygen requirement without new chest X-ray changes.\n\nSame day laboratory investigations confirmed severe haemolytic anaemia - haemoglobin (Hb) had dropped from 127 g/L on admission to 45 g/L, with new hyper-bilirubinaemia (96 μmol/L, normal <21) and elevated Lactate Dehydrogenase (LDH) (1806 U/L, normal <250). Haptoglobin, which binds free haemoglobin in haemolysis, was undetectable at <0.1g/L (normal 0.3 – 2.0). Subsequently, she developed a reticulocytosis (145 × 10^9/L, normal range 20–110). A direct Coomb’s test was strongly positive, confirming the presence of red blood cell directed antibodies. A peripheral blood smear was negative for schistocytes and a clotting screen remained normal, thus excluding thrombotic microangiopathic processes, including disseminated intravascular coagulation and thrombotic thrombocytopenic purpura. Other investigations excluded other causes of haemolysis, including paroxysmal nocturnal haemoglobinuria, mycoplasma induced haemolysis and serum electrophoresis for lymphoproliferative disorders.\n\nA putative diagnosis of Ceftazidime-induced DIIHA was made. Initial management included stopping the IV antibiotics and transfusing 2 units of packed red cells with parenteral steroid cover. Subsequently, oral steroids (Prednisolone 50mg/day) were commenced and continued until induction of remission. Once remission was achieved her steroids were to be tapered. As chronic haemolysis can induce folate deficiency, IV Folic Acid was also initiated. She improved significantly both clinically and biochemically, with her Hb recovering to 100 g/L within 10 days.\n\n\nDiscussion\n\nDIIHA is rare, under-recognised, and underdiagnosed, and data supporting it is poor. Often, only dramatic haemolysis leads to appropriate investigations2. DIIHA is commonly associated with the presence of drug-independent and dependent antibodies.\n\nDrug-independent antibodies are true RBC autoantibodies, targeting RBC components and not the offending drug. It is postulated that such offending drugs affect the immune system to cause RBC autoantibody formulation and subsequent haemolytic anaemia but the exact sensitization mechanism remains unknown2.\n\nDrug-dependent antibodies result from immune sensitization and generation of antibodies directed at epitopes on the drug and/or its metabolites, or a combination of drug and RBC membrane2,4. Specialized serological testing can confirm the diagnosis of DIIHA by demonstrating drug-dependent antibody induced haem-agglutination in vitro in the presence of the offending drug4. The identification of drug-dependent antibodies could be of clinical significance, as there is evidence suggesting that patients receiving a second course of the precipitating drug or closely related drugs may develop a more severe, even fatal haemolytic anaemia due to cross-reactive antibodies1,2. Hence, patients with cephalosporin-induced haemolytic anaemia should never receive further cephalosporins.\n\nClinical manifestations of DIIHA lie on a spectrum ranging from mild to severe haemolysis, with life threatening complications including acute renal failure, disseminated intravascular coagulation and shock3. Early identification and instigation of appropriate treatment is essential. The keystone of management is the discontinuation of the precipitant drug. Glucocorticoids (1–1.5 mg/kg/day of prednisolone or equivalent in adults) are commonly employed in inducing remission via reduction in antibody production.\n\nOur patient’s DIIHA occurred following the 10th dose of ceftazidime during her admission: this was her second course of this drug within two years. Rapid resolution of her haemolytic anaemia on cessation of ceftazidime with a high index of suspicion pre-treatment made serological testing for ceftazidime dependent antibodies unnecessary.\n\nThis case highlights the need for CF clinicians and microbiologists to be aware of DIIHA and its potential manifestations in order to facilitate early recognition and prompt management.\n\n\nConsent\n\nWritten informed consent for the publication of this case report was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "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\nArndt PA, Garratty G: Cross-reactivity of cefotetan and ceftriaxone antibodies, associated with hemolytic anemia, with other: cephalosporins and penicillin. Am J Clin Pathol. 2002; 118(2): 256–62. PubMed Abstract \| Publisher Full Text\n\nGarratty G: Drug-induced immune hemolytic anemia. Hematology Am Soc Hematol Educ Program. 2009; 2009(1): 73–9. PubMed Abstract \| Publisher Full Text\n\nChen F, Zhan Z: Severe drug-induced immune haemolytic anaemia due to ceftazidime. Blood Transfus. 2014; 12(3): 435–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChambers LA, Donovan LM, Kruskall MS: Ceftazidime-induced hemolysis in a patient with drug-dependent antibodies reactive by immune complex and drug adsorption mechanisms. Am J Clin Pathol. 1991; 95(3): 393–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "33284", "date": "24 Apr 2018", "name": "Jamie Duckers", "expertise": [ "Reviewer Expertise Cystic Fibrosis", "bronchiectasis" ], "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 case report details a patient with cystic fibrosis, chronically colonised with pseudomonas aeruginosa, receiving intravenous ceftazidime as part of their treatment for an acute exacerbation who develops drug induced immune haemolytic anaemia.\nCeftazidime is often used as part of the first line intravenous antibiotic regime in patients with cystic fibrosis and those with bronchiectasis who culture pseudomonas aeruginosa. This report highlights a potentially life threatening effect of this commonly used drug for which there are no firm data on frequency but haemolytic anaemia is listed on the summary of product characteristics. I would postulate that events of drug induced immune haemolytic anaemia are under reported. It is important that clinical teams remain alert to the potential undesirable effect of this well used drug, particularly as many patients may be administering ceftazidime themselves as part of home intravenous therapy regimes to treat their acute exacerbations.\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": "33283", "date": "26 Apr 2018", "name": "Charlotte Addy", "expertise": [ "Reviewer Expertise Cystic Fibrosis", "Bronchiectasis" ], "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 succinct and well written case report highlighting a rare but important complication of a first line anti-pseudomonal intra-venous antibiotic used commonly in Cystic Fibrosis care. It is possible this complication is under reported and without the appropriate investigations highlighted in this case may also be under-diagnosed.\nThe potential additions to this case are minor. In the introduction it would be useful to clarify the details of the previous four reported cases; as it is slightly unclear whether all four cases were in individuals with CF or across other diseases without tracking down each reference. Within the discussion it would also add to the clinical relevance of the case to highlight whether drug dependent antibodies show any evidence of cross-reactivity with other similar antibiotics as is seen with hypersensitivity reactions. If there is no evidence in this regard it would be useful to state this.\n\nOtherwise I am in agreement with the previous reviewer that clinical teams are alerted to this potentially significant adverse reaction.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-475
https://f1000research.com/articles/7-245/v1	28 Feb 18	{ "type": "Research Article", "title": "Identification of publicly available data sources to inform the conduct of Health Technology Assessment in India", "authors": [ "Laura Downey", "Neethi Rao", "Lorna Guinness", "Miqdad Asaria", "Shankar Prinja", "Anju Sinha", "Rajni Kant", "Arvind Pandey", "Francoise Cluzeau", "Kalipso Chalkidou", "Neethi Rao", "Lorna Guinness", "Miqdad Asaria", "Shankar Prinja", "Anju Sinha", "Rajni Kant", "Arvind Pandey", "Francoise Cluzeau", "Kalipso Chalkidou" ], "abstract": "Background: Health technology assessment (HTA) provides a globally-accepted and structured approach to synthesising evidence for cost and clinical effectiveness alongside ethical and equity considerations to inform evidence-based priorities. India is one of the most recent countries to formally commit to institutionalising HTA as an integral component of the heath resource allocation decision-making process. The effective conduct of HTA depends on the availability of reliable data.\n\nMethods: We draw from our experience of collecting, synthesizing, and analysing health-related datasets in India and internationally, to highlight the complex requirements for undertaking HTA, and explore the availability of such data in India. We first outlined each of the core data components required for the conduct of HTA, and their availability in India, drawing attention to where data can be accessed, and different ways in which researchers can overcome the challenges of missing or low quality data. Results: We grouped data into the following categories: clinical efficacy; cost; epidemiology; quality of life; service use/consumption; and equity. We identified numerous large local data sources containing epidemiological information. There was a marked absence of other locally-collected data necessary for informing HTA, particularly data relating to cost, service use, and quality of life. Conclusions: The introduction of HTA into the health policy space in India provides an opportunity to comprehensively assess the availability and quality of health data capture across the country. While epidemiological information is routinely collected across India, other data inputs necessary for HTA are not readily available. This poses a significant bottleneck to the efficient generation and deployment of HTA into the health decision space. Overcoming these data gaps by strengthening the routine collection of comprehensive and verifiable health data will have important implications not only for embedding economic analyses into the priority setting process, but for strengthening the health system as a whole.", "keywords": [ "Health Economics", "Health Technology Assessment", "India", "data" ], "content": "Methods\n\nThe information presented here is drawn from the experience of the authors in conducting and contributing to health economic analyses, including HTA, in the Indian context. We first identified the key areas of information needed for these types of analyses (see Figure 1). We then used our own experience to document data sources in each of these areas. The authors also approached key informants working within the broad field of evidence science in India for additional inputs and to ensure that the information presented here was sufficiently comprehensive. Finally, each source of data was assessed for its ability to fulfil the needs of HTA. Although, this is not intended to provide an exhaustive account of all databases available across India, this should offer a starting point for researchers wishing to engage in HTA using data from India.\n\nKey: QOL = quality of life; Epidem. = epidemiology.\n\n\nResults\n\nTo construct an economic model for HTA that includes both the costs and impact of a health care programme or intervention within a particular context, data in a range of key areas is needed. We have grouped data needs from these areas into the following categories: clinical efficacy; cost; epidemiology; quality of life; service use/consumption; and equity.\n\nInformation on clinical efficacy is necessary in order to understand whether a given intervention is doing what it was intended to do, and how well it achieves this, compared to all reasonable comparators. This is primarily established through randomised controlled trials (RCTs).\n\nIn the area of costs, data is needed on the cost of providing a service, or set of services, to providers and patients in order to conduct a comprehensive economic evaluation to determine cost effectiveness. Cost data is outlined in this paper from a health provider and healthcare perspective16,19, as recommended by the reference case for economic evaluation by the international decision support initiative (iDSI;20). This covers the direct patient and family costs related to the purchase of healthcare, as well as the costs borne by healthcare providers.\n\nCountry-specific epidemiological data is what allows researchers to contextualise an economic model and ensure that any conclusions drawn from the results of the model are appropriate for that given context. Epidemiological information includes demographics, vital statistics and burden of disease data. Demographics refers to quantifiable characteristics of a given population, such as gender, geographic location, or socio-economic income status. Vital statistics are information relating to births and deaths of a population, including age at death and the cause. Burden of disease refers to the impact of a health problem on a population, and can be measured by financial cost, mortality, morbidity, or other indicators such as quality adjusted life years (QALYs) or disability adjusted life years (DALYs). Together, this data underpins a large part of what constitutes an economic evaluation model.\n\nService use data provides information regarding how many people are using a particular service or seeking care which requires a certain intervention. This estimated uptake data is used alongside epidemiological data regarding disease prevalence in economic models to estimate the population impacted by an intervention or service. It is also used in estimating the costs of a particular intervention or health care activity and to inform estimates of the likely budget impact of introducing a new intervention into the health system.\n\nHealth Related quality of life data is used to calculate how a given intervention affects quality of life (QoL), and calculate associated quality adjusted life years (QALYs). QALYs are a metric used to estimate an incremental cost effectiveness ratio (ICER), and are the most commonly used metric to represent the impact a therapy has on the length of life while also taking into account any changes in the health-related QoL. QoL datasets are comprised of two separable components – a comprehensive set of health state information based on stated preferences to each QoL dimension, and a weighted utility value generated for that given health state21. The latter component is used to calculate QALYs. It is also possible to utilise burden of disease data, in the form of disability adjusted life years (DALYs), as an alternative to the QALY to capture the disability associated with living with a given condition, and the alleviation of disability after an intervention, in an economic analysis.\n\nData regarding equitable and equal access, and utilisation of services is essential to allow for ethical information to be considered in the decision-making process alongside evidence of cost effectiveness. In the Indian context, this may relate to inequity as a consequence of gender, caste, religious beliefs, age, geographic location, socio-economic status22–24.\n\nFurther information for each data source is provided below, and summarised in Table 1.\n\nTable 1 key: GFHS, Government Financed Health Insurance Schemes; HTA, Health Technology Assessment; ICMR, Indian Council of Medical Research; MHFW; Ministry of health and family welfare; MRD, ministry of rural development; MS, ministry of statistics;; MHA, Ministry of Home Affairs; OOP, out of pocket spending; SRS, sample registration system; WHO, World Health Organisation.\n\nNo formal data quality assessment was carried out by the present authors. However, the Niti Aayog, in collaboration with the Ministry of Health and Family Welfare and the World Bank, have recently published a comprehensive India health index report25, which draws from many of the same data sources outlined below, including the National Family Health Survey and the Health Management Information System portal data, where a thorough quality assessment was undertaken. The authors cite poor data quality as a major limitation of their health index estimates, highlighting a need for urgent improvement in the capture of health and demographic data and the adoption of robust data quality mechanisms across India. As such, all researchers undertaking economic analyses in India should interpret secondary data with caution, and triangulate multiple sources where possible to better understand the data reliability.\n\nRobust HTA depends on reproducible and verifiable insights into the clinical effectiveness of a given health intervention17,19. Indeed, it is impossible to establish whether a technology is cost effective if clinical efficacy is unknown or ill-established. Clinical efficacy data is generally available in the published literature for interventions that are presently available in the market. Large RCTs are the optimal source of clinical efficacy data, and these should inform clinical outcome estimates wherever possible. Systematic reviews and Meta analyses of RCTs are the gold standard for deriving clinical outcome and safety metrics. In the case of novel technologies, outcome data may be provided by manufacturers and industry and should be interpreted with caution. The Central Drugs Standard Control Organization (CDSCO) is the National Regulatory Authority in India and publishes a list of approved pharmacological interventions and their indications in India, which is updated regularly. All clinical trials in India are registered in the Clinical Trials Registry of India (CTRI), which is a primary register connected to the International Clinical Trials Registry Platform (ICTRP), an international registry run by the World Health Organization, which houses global data on clinical trials26. The Indian Council of Medical Research (ICMR) is the apex body responsible for the formulation, coordination and promotion of all biomedical research in India, and is a signatory to the WHO joint statement on clinical trials, which mandates that all clinical trials that they fund, co-fund or support would provide public disclosure of results within a year of trial completion27.\n\nThe Cochrane Library is the largest international repository of systematic reviews and meta-analyses and should be searched as a first port of call for any pooled statistics regarding clinical efficacy. The ICMR has established an Advanced Centre for evidence-based medicine that hosts the South Asian Cochrane Network and Centre at the Christian Medical College, Vellore, and has procured a national subscription to The Cochrane Library to ensure that it is accessible to scientists across India28. Researchers should conduct a thorough quality appraisal for all evidence used in an economic analyses, using checklists such as CASP or GRADE. The Cochrane Collaboration provides a useful pathway and tutorial for analysts to use to guide approaching grading the quality of evidence. Quality of the data inputs should be transparently reported and taken into consideration when using the evidence from the evaluation to inform decision making.\n\nThere may be circumstances where clinical efficacy data should be locally sourced, such as for certain technical surgical interventions, diagnostics that require skilled interpretation, and public health programs that are highly context-specific. Outcomes of these interventions may vary based on training and knowledge of practitioners, and availability of adequate infrastructure and equipment. Researchers should seek expert clinical advice regarding sources of data and appropriateness of transferring international data for use in economic evaluations to inform decision making in India.\n\n\nEpidemiological data\n\nThe government of India collects epidemiological information periodically through the Health Management Information System (HMIS) and other varied survey sources such as National Family Health Survey (NFHS), District Level Household Survey (DLHS; for reproductive and child health information), and Sample Registration System (SRS) census data. The Most recent round of the NFHS, also known as the District House Survey, data (2015–2016) is the most comprehensive source of primary demographic data in India, where information was collected from over 600,000 households. The Niti Aayog, in collaboration with the Ministry of Health and Family Welfare and the World Bank, have recently published a comprehensive India health index report25, which draws on each of the aforementioned data sources to compile a set of health index statistics for each state and union territory across India. This report highlights key information such as neonatal mortality rate, under-five mortality rate, full immunization coverage, institutional deliveries, and rates of both communicable (TB and HIV) and non-communicable diseases across each State and Union territory.\n\nBurden of disease refers to the impact of a health problem on a given population. An important new source of disease burden data for India has recently been published by the Indian Council of Medical Research (ICMR) and the Public health Foundation of India (PHFI), in collaboration with the Institute for Health Metrics and Evaluation (IHME29). This study collected, collated, and synthesized census information, vital registration statistics, Sample Registration System information, large scale national household surveys, cohort studies, disease surveillance data, disease-programme-level data, and administrative records of health services and disease registries in order to estimate disease burden in every state and union territory of India. This marks an important step forward in terms of data availability at the state-level for burden of disease and will provide an invaluable resource for researchers undertaking economic analyses in India.\n\nFurther disease-specific epidemiological data is generally available for most conditions in the published literature, though availability varies according to condition. Much of the epidemiological data regarding infectious and communicable disease in India is captured by the integrated disease surveillance program, a Ministry of health and family welfare department under the directorate general of health services. There are also a number of condition-specific programs within the Ministry of Health and family welfare which collect condition-specific information, such as the National program for the control of blindness, or National Programme for Prevention and Control of Cancer, Diabetes, Cardiovascular diseases and Stroke (NPCDCS). The ICMR also runs a network of 26 disease specific institutes across India, such as the Antimicrobial Resistance Surveillance Network, or the Rotavirus Surveillance Network, which collate data on clinical, epidemiological, and virological information, which is used to devise evidence-based treatment guidelines to improve care and monitor and evaluate transmission-modifying interventions such as vaccines. Researchers conducting a HTA study in India should consult the Ministry of Health and Family Welfare website, and individual ICMR institute websites (e.g. National institute of Research in Tuberculosis or National Institute of Malaria Research – see the ICMR website for a full list of institutes) to check whether additional epidemiological information is available for their clinical area of interest.\n\nThe Sample Registration System, part of the government census program, is mandated to collect verbal autopsy data in India. However, the completeness and quality of vital registration data for mortality in India has been highlighted as inadequate, where a recent assessment of the national civil registration and vital statistics systems in place found that available data was incomplete and of poor quality, with a medically-certified cause of death certificate recorded in only ~16% of the total death registered30. In order to address this paucity of reliable data, the ICMR will build on its previous efforts to capture cause of death in 200931, and undertake a thorough verbal autopsy study in early 2018. The global collaborative Million Deaths Study, which has been commissioned by the Registrar General of India since 2001, has also collected data in 1.3 million homes in more than 7000 randomly selected areas of India, and provides a useful resource for estimation of mortality data in India32,33.\n\nHigh healthcare costs combined with low social health insurance penetration has resulted in high out-of-pocket (OOP) expenditure for people across the country, where OOP spending makes up over 80% of health spending in India and represents a major contributor to financial impoverishment in the country29,30. OOP therefore represent a critical component of the costs of health care delivery. Additional costs such as costs to other sectors and wages forgone are not covered in this paper. Data for OOP spending was collected as part of the most recent National Sample Survey Office (NSSO; 2014), under the ministry of statistics. Cost data captured by the NSSO relates to expenditure on medicine; reluctance to seek medical services due to financial constraints; reliance on borrowings for medical expenditure; coverage by a health insurance program; expenditure on institutional childbirth; expenditure on hospitalisations and non-hospitalised treatment. In the NSSO survey, OOP can be broken down by broad health condition but are not disease specific. A number of primary research studies have also been published which provide additional data on OOP at the district level across numerous states, and these can be used to complement NSSO.\n\nService delivery data covers cost of all activities related to the provision of healthcare; including medical and allied health professional staff costs, and unit costs of equipment and infrastructure utilised during the delivery of care. Cost data is generally unavailable for service and unit indices, and primary data collection is required in most instances. Primary research has been undertaken in isolated instances to capture service delivery costs for a specific condition or level of care within a given district, however such data is very limited.\n\nThe World Health Organisation India country office has recently undertaken a comprehensive exercise to collate information on the packages of services covered by 22 different Government funded Health Insurance Schemes across the country, and their service rates. This is the first exercise of its kind to bring together information form myriad schemes across the country into a single database. This information can be used to compare coverage of services across different packages, and the rates at which those services are delivered. This database provides both package rates averaged across schemes by state and disaggregated by individual packages. This provides a highly useful starting point for any researcher undertaking a cost effectiveness analysis in understanding coverage and price of specific services, providing that those services are covered by any government funded scheme. However, it must be noted that these estimates are reflective of tendering price and thus cannot be assumed as accurate reflections of true cost.\n\nIn light of the general paucity of cost data , particularly in relation to disease-specific cost information and disaggregated estimates for individual units costs in India, a large primary costing study has been undertaken by PGIMER Chandigarh, supported by the international Decision Support Initiative, where provider cost data has been collected from a sample of over 200 health facilities across 6 states, covering all levels of the health system (more information is available online). Once finalised, this data will be made freely available in the form of an online database resource. The aim of this database is primarily to inform the HTA process, and all researchers across India will be able to use its contents to generate unit cost data to inform their analyses.\n\nInsurance claims data also provide an important source of information on the cost of health care in India. Insurance claims databases contain rich data on secondary care (hospital) claims for individual procedures, however, this data is not routinely made publicly available and reflects the tendered price of healthcare, rather than the actual cost of procuring and providing a given service. There are isolated databases of such information, such as reimbursement rates for the Central Government Health Scheme, or rates of services covered under RSBY. Should this kind of data be made more widely available for public access and scrutiny in future, it would provide an insight into the financing of healthcare in both the public and private sectors, which could help inform more reliable economic models.\n\nAt the global level, The WHO has also recognised the lack of reliable national cost data, and developed two tools to support national costing studies: WHO Cost effectiveness and Strategic Planning (WHO CHOICE) and the WHO OneHealth tool. The former provides country level estimates of unit costs for inpatient and outpatient services for the public and private sectors, but is now largely outdated. The latter attempts to equip policymakers and health service planners with a framework for informing scenario analysis, costing, health impact analysis, budgeting and financing of strategies for all major diseases and health system components. However, it requires local level data to inform these scenarios. At the diseases-specific level, the Global Health Cost Consortium (GHCC) provides an additional tool to improve resources to estimate the costs of TB and HIV programs through encouraging greater transparency and standardisation in costs data collection methods. The GHCC has also developed a unit costs study repository which provides a comprehensive database of a vast array of unit costs by country, including India, related to delivery of HIV and TB services.\n\nHealth Related quality of life data is used to calculate how a given intervention affects quality of life. The European Quality of life 5 dimensions (EQ5D) is the most commonly used generic QoL measure, and has become the cornerstone of HTA in many countries. No comprehensive national dataset for QoL has been collected for India, however, a small number of studies have collected condition-specific EQ5D data34. Researchers undertaking HTA in India who wish to generate QALYs will be required to collect condition-specific EQ5D data to inform their analyses. In the absence of a national QoL tariff for India, utility weights (necessary to calculate a QALY) will need to be transferred from an appropriate existing source, for example that of other countries in the region such as Thailand (see the EuroQol webpage for a list of all published country value sets). It is important to note that there are issues with transferring EQ5D datasets across countries, which can be explained by both methodological differences as well as socio-cultural differences between countries, and researchers should keep this in mind when using international tariffs to estimate local QoL data35.\n\nAt the global level, the Global Burden of Disease study (GBD) has collected world-wide data to calculate country-specific burden of disease estimates, including for the Indian population. Burden of disease can then be used to calculate DALYs, whereby one DALY equal the sum of number of years of life lost prematurely (YLLs) and a weighted measure of years lived with disability due to disease or injury (YLDs). YLL uses the life expectancy at the time of death, which can be obtained from the Sample Registration System Census database of vital statistics (2011). YLD is determined by the number of years disabled weighted by level of disability caused by a condition or disease, where the disability weights are derived from the GBD data. Many LMIC have taken to using GBD study estimates to inform economic analyses in the absence of locally-generated quality of life data. The limitations of such an approach should be noted, where the DALY does not easily allow for the modelling of different disease states, and the value judgements used to weight DALY estimates are those of international, rather than local, experts.\n\nService use data is collected in 2 ways: Through census surveys and household questionnaires; and directly through service providers.\n\nThe National Family Health Survey (NFHS), and the National Sample Survey Office (NSSO) census surveys are the largest surveys in India, and collect individual responses regarding consumption of both health and non-health services across the country. Special attention was paid to the collection of health-related information in the 2014 NSSO survey at the request of the Ministry of Health and Family Welfare, where the first national-level information was collected on rates of hospitalisation, medical care received as in-patient in medical institutions, the broad disease areas for which such medical care was sought, the extent of use of Government hospitals, and out of pocket expenditure on medicines and other health-related products incurred was also collected. Expenditure incurred on treatment received from public and private sectors was also accounted for.\n\nWhile healthcare use data is typically collected by insurers, insurance schemes do not routinely publish this data for public access. Several State-level insurance schemes do publish high-level claims data and report figures on service use, for example Aarogysari scheme in Telangana publishes their annual claims data online each year. Third Party Administrators process most insurance-related claims in India, and hold data regarding institutional-level service use, however this data is also not easily available and not routinely released.\n\nThere are a number of sources of information across the country that provide information on equity, and a robust analyses of equity information to inform a given HTA should take into account multiple data sources in order to provide a comprehensive assessment of this domain. Data may be extrapolated from large survey samples, such as the National Family Health Survey (NFHS), and the National Sample Survey Office (NSSO), and Health Management Information System portal, where data is available which links demographic information such as gender, geographic location, urban or rural dwelling, age, and socio-economic status to health behaviours and experiences. The Socio-economic and Caste Census was also conducted under the ministry of Rural Development in 2011, which provides additional comprehensive information on these two social determinants of health. Data published by government insurance programs at both the national and state level, such as RSBY, may also be a useful source of information regarding equity in the Indian context, where a number of published studies highlight inequitable access to social health insurance36,37. The recently published Global Burden of Disease India profile (201729), and Niti Aayog ‘Health States’ report25 further highlight health inequity between states across the country and could also be used to inform equity analysis for the conduct of future HTA studies.\n\nIndia lies at a critical juncture, where political discourse regarding Universal Health Coverage has reinvigorated interest in improving the way in which healthcare is financed, purchased, provisioned, monitored, and governed. The institutionalisation of HTA for setting evidence-based priorities, and the associated strengthening of a robust network of routinely collected and verifiable health data will be a vital lever for systemic reforms to transform India’s fragmented healthcare system. The World Health Organisation urges member states to develop and improve the collection of data on health intervention and technology assessment as part of their Health Intervention Technology Assessment Resolution (20144). If the government is committed to leveraging HTA as a tool for realising UHC, health information systems and the platforms for collecting health data will need to be strengthened and adequately governed.\n\nStrengthening data collection should begin at the clinical trial phase, where protocols can be put in place to ensure that cost, safety, efficacy, and socio-demographic information are collected from the earliest phases of technology development. In European countries where it is common process for technologies to go through a process of HTA before entering the market, such data is routinely collected by industry bodies in the development and piloting phases5. Improving the capture of data on health technologies in India, where the health technology industry is one of the largest and most powerful globally, would have important ramifications beyond India for the global health innovation market.\n\nAs a Nation leading global advances in technology, telecommunications systems, and mobile devices, there is significant opportunity for India to leverage technological innovation to support health system strengthening. The rollout of Aadhar across the country, which provides each citizen with a unique and identifiable digital identity, gives rise to the possibility of linking health related information to individuals, and consolidating such data for macro-level surveillance and real-time quality improvement. The combination of the these unique digital identifiers and the ubiquitous access to technology further afford the opportunity to aggregate data, expertise, and experience from a variety of sources for health-focused problem-solving a learning health system. Working with large datasets enhances the ability to detect and monitor quality improvement, and can thus meet both clinical and business needs simultaneously. This is of particular importance in the Indian context, where the government relies on harnessing the power of the private sector through mutually beneficial public private partnerships to provide access to high quality publicly-funded healthcare.\n\nIt must be noted that much of the data described in this paper is derived from and government-sponsored information systems and census surveys, where specific objectives are present and thus the presence of bias cannot be discounted. Discrepancies between administrative data and independent household surveys from around the world suggest official statistics can systematically exaggerate development progress38, and Indian datasets are not immune to such criticisms39. As India transitions away from its reliance on large-scale international donor-funded studies towards locally-sourced datasets, additional measures for safeguarding data quality and protecting against perverse incentives and the political economy of inaccurate indicators will need to be put in place.\n\nThe improved accountability inherent in making health data more transparent can have important indirect consequences in measuring and improving quality of service provision, and reducing low value care40. The ratification of the Indian Clinical Establishment Act in 2010 gives leverage to the government and private citizens alike to demand greater accountability of care across both the public and private sectors, and marks an important step forward towards more transparent health information provision. Improving the way health information is captured, reported, and utilised across the country benefits the entire population: the public are empowered to make informed choices regarding their own health, academics are afforded the opportunity to identify bottlenecks and potential levers for quality improvement, and policy makers are equipped to make more robust, defendable, and evidence-based health policies for improving the health of the population.\n\n\nConclusions\n\nThe Government of India has committed to institutionalising Health Technology Assessment as an integral component of health resource allocation decisions. This provides a comprehensive framework from which to assess the availability and quality of local evidence, and look to the future of health information capture in the Indian healthcare system. There is significant opportunity for India to leverage the dual benefits afforded by a growing commitment by the government to universalise health coverage, and a burgeoning local industry of health technology innovation. By taking advantage of the digital revolution sweeping the country and mobilising the existing cadre of data generators to strengthening the way in which information is collected, reported, and utilised, there is a unique opportunity for India to transform its health system and lead a global health data revolution.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nBill and Melinda Gates Foundation [OPP1087363].\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\nChalkidou K, Glassman A, Marten R, et al.: Priority-setting for achieving universal health coverage. Bull World Health Organ. 2016; 94(6): 462–467. 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Front Public Health. 2016; 3: 282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSandefur J, Glassman A: The Political Economy of Bad Data: Evidence from African Survey & Administrative Statistics. Centre Global Development [Internet].2014. Publisher Full Text\n\nRajan SI, James KS: Third National family health survey in india: issues, Problems and Prospects. Econ Polit Wkly. 2008. 43(48). Reference Source\n\nMorton M, Nagpal S, Sadanandan R, et al.: India’s Largest Hospital Insurance Program Faces Challenges In Using Claims Data To Measure Quality. Health Aff (Millwood). 2016; 35(10): 1792–1799. PubMed Abstract \| Publisher Full Text" }	[ { "id": "31325", "date": "16 Mar 2018", "name": "Malaisamy Muniyandi", "expertise": [ "Reviewer Expertise Health economics", "public health", "health technology assessment", "tuberculosis", "tribal health", "implementation and operational research", "demography" ], "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 focussed a very important issue in the context of Health Technology Assessment in India. Data availability and accessibility from both public and private sources is a big challenge while conducting HTA in any discipline. The authors have appropriately described their valid experiences in this article. The information provided in this article will be of use for practitioners of HTA in India.\nI recommend the authors to consider the following issues and address them:\nAuthors can consider the programmatic data which is available. For example Revised National Tuberculosis Control Programme had collected valuable patient wise data and programmatic management data including resources/technologies invested and used. Similarly other programmes like National AIDS Control Programme need to be looked into for data sources.\n\nData available with non-government organisations which have been working on various health related interventions need to focused on.\n\nData from academic institutions like IIT and universities where students and faculties are engaging in innovations and newer technologies need to be explored.\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": [ { "c_id": "3589", "date": "18 Apr 2018", "name": "Laura Downey", "role": "Reader Comment", "response": "We thank the reviewer for their insightful comments. In order to address the specific comments made by the reviewer, we have made an amendment to include the following paragraph under service-use/ consumption:Data regarding uptake of services for particular conditions is also collected by government-sponsored vertical programs, such as the National Aids Control Organization (NACO), and the National Program for the Control of Tuberculosis. HTA researchers should consult the Ministry of Health and Family Welfare website, and individual program websites for their topic area of interest to check whether additional service use information is available.We endeavored to explore the additional data sources suggested by the reviewer, including ‘Data from academic institutions like IIT’, however we were unable to identify publicly-available HTA-relevant data sources from these institutions. As such, we have not made reference to these sources in the paper amendment. We hope this is acceptable to the reviewer. We would also like to clarify that throughout the paper we refer to the ‘public sector’, meaning government-sponsored institutions, and the ‘private sector’, of which we mean all non-government organisations, including private not-for profit and private for-profit institutions. We hope that this addresses the reviewers point regarding referring to the data captured by non-government organisations." } ] }, { "id": "31324", "date": "10 Apr 2018", "name": "Rakesh Kumar Srivastava", "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 present article is the first research/review article on data availability for HTA in India. It has attempted to capture the gaps in the availability of data – in details, scope, and quality & inter state and national comparability, so that it can be used as base line to initiate HTA for major public health interventions. Since it is not a standard research article, its result have been quoted under the sub-head of need, quality, efficacy, safety, epidemiology, disease burden, cost, quality of life, service use and equity. It also suggest for taking advantage of digital Revolution…..of data generators to strengthening the way in which the information is collected, reported and utilized. A suggestion for future research is also included in the article.\n\nThe conclusion mentioned “towards commitment of GOI for institutionalizing HTA as integral component of health resource allocation decision”. With this conclusion from the research article, the relevance of details of Data Needs as given on page 4, becomes conclusive and unidirectional when it writes that “the cost data is outlined …..by the international decision support initiative (iDSI)”. Point of improvement are required for areas where suggestions has been given for iDSI which can be seen under the head of data need and service delivery cost. It is suggested to incorporate others if available without specific mention of 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? 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": "3590", "date": "18 Apr 2018", "name": "Laura Downey", "role": "Reader Comment", "response": "We thank the reviewers for their insightful comments. In order to address the specific comments made by the reviewers, we have made the following amendments: Restructured the costing section of the paper by splitting the section into patient expenditure and service delivery costs. Amended the introduction to the cost database paragraph to reflect the fact that no single cost database of this kind exists in India, and hence there is an important need to develop this kind of publicly available resource. The cited database that is presently being developed by PGIMER Chandigarh is intended to fill this important resource gap, and stimulate further research in this area in India. Restructured the conclusion to clearly summarise the key findings of the paper and their significance to the institutionalisation of HTA in India We would like to clarify that the reference to the international decision support initiative (iDS) was highlighted because of the research that this consortium supports in the area of HTA in India. We have removed the specific reference to this as per the reviewer's request so as not to confuse readers by singling out a single initiative." } ] } ]	1	https://f1000research.com/articles/7-245
https://f1000research.com/articles/7-469/v1	17 Apr 18	{ "type": "Research Article", "title": "A study on the effect of Haruan fish extract (Channa striatus) on wound healing and quality of life of coronary artery bypass grafting (CABG) patients: A prospective, double-blind, randomized, controlled trial", "authors": [ "Ahmad Farouk Musa", "Jeswant Dillion", "Mohamed Ezani Mohd Taib", "Alwi Mohd Yunos", "Saringat Baie", "Rusli Bin Nordin", "Jeswant Dillion", "Mohamed Ezani Mohd Taib", "Alwi Mohd Yunos", "Saringat Baie", "Rusli Bin Nordin" ], "abstract": "Background: Wound healing remains a primary problem in all surgical cases especially so when the length of incision is very significant as with cardiac bypass patients. The main objective of this study is therefore to assess the effect of Haruan fish extract (Channa striatus) on chest and leg wounds post-coronary artery bypass grafting (CABG) surgery with the optimum and standard patient care in two groups of randomized patients. Methods: This is a randomized, double blind clinical trial being conducted at the National Heart Institute, Kuala Lumpur. Two randomized groups of similar demographic and co-morbid histories planned for CABG were enrolled into the study. Both groups were blinded to the capsules being given to them pre- and post-operatively. Assessments were also made on wound pain, mobilization and on the health-related quality of life (HRQOL) of patients using the Nottingham Health Profile (NHP).\n\nResults: The group that received Haruan capsule showed better would healing objectively. They had better pain scores, though there was no significant difference in terms of mobilization. Overall, the HRQOL in the study group showed improved quality of life.\n\nConclusion: Our study shows the superior effect of using Channa striatus, a local Haruan fish which is easily processed into capsules in promoting wound healing, reducing pain via its anti-nociceptive effect and improving quality of life of patients after coronary artery bypass grafting surgery. It is inferred that a faster recovery from surgery confers an advantage in terms of resources to overall economic benefits. Reduction in the percentage of wound infection also resulted in reduced hospital cost. All these factors could lead to the successful commercialization of Haruan as a nutraceutical product. Trial registration: The trial was conducted from January 2012 until August 2014 and the trial number as registered with the National Medical Research Registry is NMRR-17-360-34772 (Registered 13/03/2017).", "keywords": [ "wound healing", "quality of life (QOL)", "coronary artery bypass grafting (CABG)", "Channa striatus" ], "content": "Introduction\n\nWound healing involves complex interaction between cells and mediators, which starts once the wound is inflicted, and continues for weeks. Healing is still a major problem in any kinds of surgery, particularly in cardiothoracic surgery. This is a surgery that involves a long incision over the sternum where delayed sternal wound healing is not an uncommon sight. Sternal wound infections are a known cause of morbidity and might require intervention from plastic surgeons. In severe cases, it could even lead to mortality.\n\nSternal wound infection is fairly uncommon as compared to leg wounds, with a frequency between 0.7%1 and 3.3%2, but reports based on extended surveillance on post-discharge cases showed incidences up to 9.7%3. As expected, the end-result of sternal wound infection include wound dehiscence, mediastinitis, pericarditis, osteomyelitis and endocarditis4 with associated mortality between 14% and 47%5–9.\n\nLeg wound infection, though not life-threatening as chest-wound, increases the overall morbidity of the patients. It is noted that there is a threefold increase in cost of managing patients with leg wound as compared to those with an uncomplicated postoperative course7. Complications such as wound breakdown, cellulitis, lymphangitis, fat necrosis, and delayed healing are known sequelae of leg wound infection. It is not surprising that the incidence has been reported to be as high as 24%10–13.\n\nWe have noticed that despite the different types of dressings being used, the rate of infection did not vary that much14–17. The reason for this seemingly indifferent incidence of wound infection is due to the fact that the healing was by primary intention17. The cause for such an infection is primarily deep-seated problem of the pre-operative and intra-operative rather than merely the superficial issue of wound dressing18.\n\nWith that understanding, perhaps a better way in promoting wound healing would be a systemic approach rather than topical; with all other factors remaining constant. Extracts of Channa striatus, locally known as Haruan (snakehead), a fresh water, air-breathing, carnivorous fish, has been proven to influence the different phases of the wound healing process19,20. Laboratory studies conducted on Sprague Dawley rats have shown that Channa striatus has antinociceptive effects that could reduce postoperative pain21–23. Channa striatus has been shown to contain all essential amino acids for wound healing, particularly glycine. Glycine is a major component of human skin collagen that acts synergistically with other essential amino acids that facilitate in tissue healing process. It also has a high content of arachidonic acid and polyunsaturated fatty acids that can promote prostaglandin synthesis, which is a very important compound that creates and removes inflammation, which is part of the healing process19. It has also been shown that Channa striatus promotes remodelling of collagen by the synthesis of inter, and intra molecular protein cross-linking, producing a marked increase in the tensile strength that accentuates wound healing24. Furthermore, the fish also has certain fatty acids that have been reported to possess anti-inflammatory activity such as stearic acid and oleic acid25.\n\nUp to this point, all the above studies that looked into the healing and nociceptive properties of Channa striatus have been on animal models. This is the first study that is conducted on human subjects. We therefore conducted a prospective randomized control trial (RCT) and assess not only wound healing and wound infection after CABG, but also the health related QOL of such patients, considering the anti-htinflammatory and anti-nociceptive effect of Channa striatus. The potential value of patenting such natural product and it’s usage on other types of surgical wounds, including laparotomy and Caesarean section; compounded with the commercial value associated, makes it even more attractive.\n\n\nMethodology\n\nLaboratory. The Haruan used in this study was locally reared in a special pond for Haruan; the sex of the fish was not determined. The depth of the pond should not be more than 4 feet for the fish to surface and breath, since Haruan is air breathing and carnivorous. Temperature is maintained around 25°C and pH must be maintained between 5.0 – 5.5 and salinity between 0 – 10 ppm. The Haruan capsules produced were derived from the flesh of the fish and were manufactured using strict good manufacturing practice (GMP) Guidelines by Major Interest Sdn. Bhd.\n\nHaruan (whole fish) are procured fresh to the laboratory. These fishes are kept at -18°C until preparation. The fishes are thawed prior to gutting and cleaning. The cleaned fishes are weighed and placed into the autoclave bin. The water mixture for autoclaving is prepared in relative to fish weight with respect to the volume of the water used. Therefore, a 1:0.5 mixture would contain 1kg of fish with 0.5 kg (500ml) of water. . No preservatives were used in the preparation of Haruan capsules.\n\nSterilization is carried out using Hiclave HVE-50 autoclave (Hirayama, Tokyo, Japan) with temperature setting of 110°C for 15 minutes. Upon completion of the sterilization, the fish are mixed and meshed thoroughly using a clean ladle, which has been wiped with 70% alcohol. This mixture is then transferred into a large lined aluminium pan with a dimension of 24 × 18 × 1 inches and dried using a 12 tray industrial oven at 60°C for 48 hours continuously.\n\nUpon completion, the sheets of crispy flakes are grinded using a comminuting mill and a hand grinder, Kimah Grinder Machine (CSJ-300, Penang, Malaysia) to ensure thin bone fragments are properly powdered. The resulting powder is mixed homogenously in a clear large plastic bag. Upon homogenous mixing, the powder is sieved using Retsch AS200 analytical sieve shaker (Haan, Germany), with woven wire mesh sieves of 850 micron aperture.\n\nThe refined powder was filled into empty hard gelatin capsules of pharmaceutical grade which is 1005 TSE/BSE produced by Nasmir Hard Gelatin Capsules Sdn. Bhd. (Catalogue number 1NH001, Pulau Pinang, Malaysia) based on bench scale filling method using Kimah electronic capsule arranging machine, Model 400-F1 (Penai, Malaysia). The empty hard gelatin capsule shells used are of size 0 and made of halal gelatin each weighing 0.08mg. The capsules were visually inspected for defect and are weighed for standard distribution of 250 mg using Sartorious analytical digital balance (Göttingen, Germany). The standardized capsules are packed into sterile bottles using a Kimah electronic counting machine, Model JB-2B (Penai, Malaysia). The bottles are inserted with pre-packed silica beads for moisture absorbance and cotton balls to prevent spillage.\n\nA randomized controlled trial (RCT) was completed with patients with coronary artery disease requiring coronary artery bypass grafting surgery were prospectively and randomly divided into two parallel groups. One group received Channa striatus capsule formulation derived from the fillet of Channa striatus and the control group received placebo (maltodextrine). The trial was conducted from January 2012 until August 2014 and the trial number as registered with the National Medical Research Registry is NMRR-17-360-34772 (Registered 13/03/2017).\n\nWe used the PS Software version 3.1.2 for power and sample size calculation. We have planned a study of a continuous response variable from independent control and experimental subjects with 1 control per experimental subject. Based on a previous study26 we planned a study of independent cases and controls with 1 control per case. Prior data indicated that the failure rate among controls was 0.027. If the true relative risk of failure for experimental subjects relative to controls is 6.9, then we needed to study 58 experimental subjects and 58 control subjects to be able to reject the null hypothesis that this relative risk equals 1 with probability (power) 0.8. The Type I error probability associated with this test of this null hypothesis is 0.05. We used an uncorrected chi-squared statistic to evaluate this null hypothesis. Assuming a drop-out rate of 20%, the minimum sample size required was about 70 (58 + 12) subjects per group. In this study, we managed to recruit 253 patients, and 183 completed the three months follow-up. We therefore surpassed the minimum requirement of sample size of 140 subjects, allowing us reach a power of study of 0.8.\n\nInclusion criteria:\n\n1. Male or female\n\n2. More than 18 years of age\n\n3. Elective, urgent, or emergency coronary artery revascularization\n\nExclusion criteria:\n\n1. Less than 18 years old\n\n2. Refusal to have surgery\n\n3. Inability to give informed consent\n\n4. Documented allergy to fish or fish product\n\nThe two randomized groups based in the National Heart Institute (IJN) were matched according to sex, age, New York Heart Association (NYHA) criteria (indicates patients’ hearts condition and the severity of their symptoms. Class I to Class IV), ejection fraction, and diabetic status. Randomization was done via simple computer-generated randomization with odd or even RN numbers in each group. Operative and peri-operative conditions were also similar for both groups. All subjects received an identical prophylactic antibiotic regime consisting of Cefazolin 2gm at induction and 1 gm 12 hourly for 48 hours. Gentamicin 2mg/kg was also given at induction.\n\nA uniform method of wound closure and disinfection protocol was followed. Skin was disinfected with Betadine followed by povidone iodine 10%. The method for wound closure was similar for both groups, namely double layer sutures up to the intra-cutaneous skin. Going from deep up to the surface, the pre-sternal fascia was closed with 1-0 Vicryl suture in a continuous type followed by closure of the subcutaneous tissue. Skin was closed with Monocryl 3/0 in subcuticular manner. For the leg wound, the subcutaneous tissue was closed with Vicryl 0, followed by continuous skin suture with Maxon 3/0.\n\nTitanium clips were used to secure branches of the internal mammary artery on the chest wall and branches of the saphenous vein in the leg. Some saphenous veins branches were ligated with Silk 3/0 instead. When closing the sternum, steel wire sutures were used.\n\nBoth groups took either, 2 capsules (500mg) of Channa striatus, or 2 capsules of placebo (maltodextrine) daily for a minimum of six weeks, starting from day one post-operatively. If the patient was still ventilated, the capsules were broken, and the content administered via a Ryle’s tube. Investigators and healthcare workers, as well as participants, were blinded to the capsules received. Only the manufacturers who supplied the drug were aware of the meaning of the label on the capsule, letter E or O.\n\nPrior to randomization of participants, the study was explained, including risk and benefits to suitable candidates, and written consent was obtained. Consenting participants were then randomized into the trial. The study was conducted with ethical approval from the National Heart Institute Malaysia (IJNEC/04/2011) and National Medical Research Register (NMRR-17-360-34772).\n\nWound Infection. Both sternal and leg wounds were inspected and assessed daily until subjects were discharged. Information at 6 weeks and 3 months postoperatively was collected from patients in the clinic or over the telephone. Each wound was scored using the ASEPSIS system (Refer to Supplementary Material 1). We realized that this is a more objective method in assessing wound infection rather than being subjective and open to biases. The end result from this scoring system will determine whether the wound is considered significant or not by obtaining a score of 21–30. If the wound is more serious with a deep seated wound infection, it will have a score of 21–30, and should there be evidence of bone infection, then the score would be >41.\n\nWound pain. A Visual Analogue Scale (VAS) of 0 to 10 cm was used to assess the degree of wound pain. A score of 0 cm represents “no pain” and a score of 10 represents “severe pain”. Pain assessment will be recorded from day 1 and will continue daily until the subject is discharged. It will be further assessed during the clinic visit at 6 weeks and 3 months postoperatively.\n\nMobilization. Similarly, a VAS of 0 to 10 cm was used to assess the degree of mobilization. A score of 0 cm represents “inability to walk” and 10 cm represents “excellent mobilization.” Assessment of mobilization was made on post-operative days 3 and 4 since there was restrictions in mobility on days 1 and 2 after surgery. Similarly, it was assessed again during the clinic visit at 6 weeks and 3 months postoperatively.\n\nHealth related quality of life. In this study, we decided to assess the health-related quality of life (HRQOL) using the Nottingham Health Profile (NHP) – Part 1. The NHP is known to be very efficient in clinical studies. It looks into parameters such as discomfort and pain apart from the general well-being of a patient. And it is found to be more objective in assessing the health status of the patients. There were 38 subjective statements which were divided into six sections on NHP Part 1 namely physical mobility, social isolation, emotional reaction, energy, pain, and sleep. Each section will have a score range from between 0–100 by adding the item weight to each answer that is deemed positive.\n\nThe questionnaire was distributed to both groups of patients before CABG and at six weeks and three months during clinic visit postoperatively. The questionnaires were administered using the questionnaire-interview approach.\n\nData was analysed with IBM SPSS Statistics version 24.0 and examined for normality using the Kolmogorov-Smirnoff test and stem-and-leaf plot. When normal, we proceeded with parametric tests; however, if not normal, we proceeded with the non-parametric tests.\n\nThe results were presented as means ± standard deviations for scaled measurements; with numbers and percentages for categorical measurements. The unpaired t-test was used to examine mean differences for wound infection, wound healing, pain, and mobilization across the two groups. Differences in proportions was examined using the Chi-square test.\n\nIn examining the mean QOL score, differences between the two groups (pre-operative, six weeks, and three months), the mixed mode two-way repeated measure ANOVA with post-hoc multiple comparison test (between and within subjects) of the two groups was performed.\n\nTo determine the factors influencing the change of QOL after CABG, with the dependent variable being binary (improved or worsened), we performed an initial simple logistic regression (SLogReg), and examined the statistical significance of each independent variable such as the amount of revascularization and duration of surgery on the outcome. We then performed a multiple logistic regression (MLogReg) including variables with a level of significance ≤ 0.20 in the multivariate logistic regression and controlling for the effects of possible confounding variables (sex, age, NYHA Criteria, ejection fraction, and diabetic status). A p value of 0.05 was taken as the level of significance.\n\nRefer to Figure 1 for the study flow chart.\n\n253 patients were recruited from January 2012 till August 2014. Only 183 patients (72.3%) completed the study up to three months follow-up. A total of 40 patients (15.8%) were lost during follow-up and 5.5% discontinued their enrolment in the study. The main reason why these patients were lost during follow-up was for a simple reason that they did not turn-up for the scheduled follow-up. This is due to the fact that IJN is the largest heart centre in the Malaysia and the patients were from all over the country. And since many were former government servants and stay in the rural areas all over the country, travelling to the city centre was not only time-consuming but expensive. Seven patients (2.8%) had their operations cancelled.\n\nThe patients were predominantly male (86.2%), the majority being Malay (86.7%), followed by Indians (15.4%) and Chinese (7.9%).\n\nThe mean weight was 71.92±13.10 kg ranging from 45–132kg and the mean BMI was 27±4.3 kgm2.\n\n125 (49.4%) of them received Capsule O (placebo) and 128 (50.6%) received Capsule E that contains Haruan extract.\n\nOut of the 246 patients who underwent CABG, 224 patients (91.06%) had an elective operation, while only 8.94% were emergency cases.\n\nOff-pump CABG was performed on only 16.7% of patients as compared to the conventional on-pump (83.3%). Mean operation time was 186.94±48.9 minutes ranging from 75 to 350 minutes. The mean bypass time was 82.08±27.55 minutes.\n\n65 patients (26.42%) developed atrial fibrillation (AF) post-operatively while 10 patients (4.07%) developed supraventricular tachycardia (SVT).\n\nOnly 10 patients (4.07%) had their chest re-opened. Known complications such as renal failure occurred in only 2 patients (0.81%) and stroke in another 2 patients (0.81%) as well. Overall, the mortality rate was 3.6% with nine patients dead.\n\nMean hospital stay was 10±11.86 days and mean ICU stay was 2.97±7.67 days. Both the duration of ICU stay (2.31±2.65), and hospital stay (9.24±8.58) was shorter for patients on Capsule E in comparison to Capsule O (3.69±10.7 and 11.1±14.6, respectively), although this was not statistically significant (p=0.170, 0.230, respectively).\n\nThe ASEPSIS score showed no statistically significant difference between Capsule E and O on Day 3, 6, 6 weeks and 3 months (p=0.243, 0.805, 0.117, 0.980, respectively).\n\nThere was no statistically significant difference in the mean pain score using the visual analogue scale (VAS) between the two groups post-operatively (Capsule E: 0.39±0.25, Capsule O: 0.43±0.24, p=0.290) and no difference on mobilization (p=0.668).\n\nAssessment of HRQOL comparing both groups showed no significant difference at discharge (p-values: emotion=0.48, physical=0.54, energy=0.16, social=0.071, sleep=0.87, and pain=0.29) and assessment at 6 weeks and 3 months follow-up showed no statistically significant difference for all six components of HRQOL except for social level at 3 months (p=0.04).\n\nRefer to Figure 2 for study results flow chart.\n\n\nLimitations\n\nThere were a few limitations to this study. The first is in getting the patients’ consent in enrolling into the study. A few patients had this misconception that Haruan would cause hypertrophic wound or sometimes even keloid. This was the misperception that they had and no amount of talking or persuasion would convince them. And of course, considering that their enrolment is voluntary, their withdrawal from the study is unavoidable. Hence there were almost 30% of patients who did not manage to complete the study. The second limitation is regarding the compliance of the patients to take the capsules provided to them. While they were on the ward, this issue of compliance did not arise, since the Haruan capsules or the placebo were served by the attending staff nurses. However, compliance was not ensured when the patients were discharged from the wards. The third is regarding how the Nottingham Health Profile was administered. We decided for the questionnaire to be conducted by the researcher and not independently answered by the patients without the presence of the researcher. This could have resulted in some bias when answering the question on NHP by the patients. While we understand that this interview mode is not free from bias, it is still the least burdensome method, which only requires the respondent to speak the same language as the interviewer, and to have a basic verbal and listening skills. Considering that almost all Malaysians speak Malay and a majority of them speak English as well, this is probably a more effective way. We believe that the interview method would allow us to clarify ambigu ous questions, and maintain the motivation.\n\n\nDiscussion\n\nWound healing has always been an obsession among surgeons. And despite the advance in surgical dressings over the years, surgeons have always been trying to find novel ways in promoting wound healing. Haruan or Channa striatus has been known amongst the locals since ancient times to promote healing, and has been used as a form of nutritional supplement. Normally the fish is consumed after it is fried, grilled or boiled; as a traditional remedy for wound healing especially after delivery of caesarean section. There, however, are no clinically randomized trials so far that have been conducted to prove or disprove this belief.\n\nOur study – the first as far as the authors are concerned - has shown the clinical benefit of Channa striatus in promoting wound healing among CABG patients. The study was conducted in a double-blind manner, and only after the results were analyzed, were the researchers informed of the content of Capsule E and Capsule O. There was no way for the researchers to know before hand of the content of the capsules.\n\nThe results scientifically confirm the long-held belief of the benefit of Haruan fish. The properties of Haruan to promote wound healing have been postulated. Perhaps it was the way in which Haruan had positive influence on the immune system in regulating the prostaglandin synthesis via the possession of ω-3 polyunsaturated fatty acids has made it effective in stimulating the healing process20.\n\nThe healing process requires both amino acids and fatty acids. And these have been found in abundance in Haruan fish. Compounds such as glycine, alanine, proline, arginine, serine, leucine, isoleucine, and phenylalanine are known to combine with aspartic and glutamic acid to form polypeptides. These form the basic foundation for healing to take place. Studies have demonstrated that these amino acids and fatty acids are present in Haruan20.\n\nSimilarly, Haruan also contains a high content of arachidonic acid, 20:4ω6. This arachidonic acid is thought to be related to the wound healing property of Haruan20. The platelet aggregation and adhesion to the endothelial tissue that initiates blood clotting is thought to be induced by arachidonic acid.\n\nResearchers at the Analytical Biochemistry Research Centre at the Universiti Sains Malaysia have done proteomic profiling on Haruan extract and found out that 25% and 26% of the total protein detected in freeze- and spray-dried C. striatus extract respectively are actin, myosin, and tropomyosin27. A recent study has also shown that actins are essential for a range of cellular functions including; the maintenance of cell shape, polarity, cell division, cytokinesis, vesicle and organelle movement, cell signaling, establishing and maintaining cell junctions, and the regulation of transcription28. And this process is postulated to take effect during the process of re-epithelialisation, which is part of proliferative phase during a healing process28.\n\nIt is also known that actin acts in synchrony with myosin in promoting wound healing. This biomechanical process in driving cell motility has previously been shown29.\n\nTropomyosin, which controls the isoform-specific regulation of diverse actin filaments, is also suggested to play a role during wound healing30. This finding is also confirmed by Lees et al31. in their report on the role of tropomyosin as a regulator of actin functioning during the process of wound healing.\n\nSo it is possible that actin, myosin and tropomyosin, which can be found in Haruan, contributed to the expedition of the wound healing process.\n\nTwo types of collagen, namely type I and type II, have also been detected in Haruan through proteomic profiling27,32. Collagen is a well-known substance that gives tensile strength during wound healing. In fact, it has been shown that collagen and other components of the extra-cellular matrix mainly formed the granulation tissue33. It is said that the granulation tissue then contracted, slowly closing the wound, and at the same time aligning the collagen fibres in the extracellular matrix33. This leads us to believe that the collagen found in Haruan promotes the maturation of granulation tissue, and hence, expedite wound healing.\n\nApart from the healing properties, Haruan has also been proven to promote the quality of life of patients’ post-CABG. The HRQOL study shows that there was significant change in the energy, pain, emotion and physical level.\n\nThese findings demonstrate the much-acclaimed “rejuvenating effect” of Haruan. It is not an exaggeration to propose that the high content of fatty acids and certain amino acids coupled with the anti-nociceptive effects34,35 might have indicated that they somehow contributed to the natural well-being of patients’ post-CABG.\n\n\nConclusion\n\nOur clinical study, though laborious, has demonstrated the effects of using Channa striatus, a local fish known as Haruan, which is easily processed into capsules in promoting wound healing, reducing pain via its anti-nociceptive effect and improving quality of life of patients after coronary artery bypass grafting surgery. This is the first randomized, double blind clinical trial being conducted on this matter although there have been anecdotal evidence on the efficacy of Haruan. It is inferred that a faster recovery from surgery confers an advantage in terms of resources to an overall economic benefit. Reduction in the percentage of wound infection also resulted in reduced hospitalization cost. We believe this will lead to the successful commercialization of Haruan, which could be reared commercially as a nutraceutical product.\n\n\nData availability\n\nDataset 1 - Health Related Quality of Life for all participants 10.5256/f1000research.13372.d19659336\n\nDataset 2 – Surgical wound healing data for all participants 10.5256/f1000research.13372.d19659437", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by Monash University Malaysia under Major Grant, CRM279.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgment\n\nWe would like to acknowledge the full cooperation of the Surgeons at the Cardiothoracic Department of the National Heart Institute, Kuala Lumpur, the nursing staff especially those of the Cardiac Intensive Care Unit, High Dependency Unit, Surgical Wards and Surgical Clinics and to the staff of the Research Department for making this study a success. We would also like to acknowledge the massive contribution of the late Prof Saringat Baie in developing the Haruan capsules. His work is now being continued by his protégé Dr Mohd Nazri Ismail at the Analytical Biochemistry Research Centre, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia. He is contactable at mdnazri@usm.my.\n\n\nSupplementary material\n\nSupplementary Material 1 – definition of ASEPSIS\n\nASEPSIS is an acronym for Additional treatment, the presence of Serous discharge, Erythema, Purulent discharge and Separation of the deep tissue, the Isolation of bacteria and the duration of inpatient Stay. Points were allotted to the ASEPSIS system for the extent of wound disturbance showing serous (0–5) or purulent (0–10) exudate, erythema (0–5), and separation of the deep tissues (0–10). Points were also added up for criteria up to three months following surgery: antibiotic treatment, 10 points; drainage under local anaesthetic, 5 points; debridement under general anaesthetic, 10 points; positive microbiology isolate, 10 points; extended hospital stay beyond ten days due to wound infection, 5 points. The total score will then be used to reflect the severity of infection as shown:\n\n0–10: satisfactory healing\n\n11–20: disturbance of healing\n\n21–30: minor wound infection\n\n31–40: moderate wound infection\n\n41: severe wound infection\n\nSupplementary Material 2 – Protocol for the current study\n\nClick here to access the data.\n\nSupplementary Material 3 – Completed CONSORT checklist\n\nClick here to access the data.\n\n\nReferences\n\nNewman LS, Szezukowski LC, Bain RP, et al.: Suppurative mediastinitis after open heart surgery. A case control study of risk factors. Chest. 1988; 94(3): 546–553. 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Publisher Full Text\n\nSonnemann KJ, Bement WM: Wound repair: toward understanding and integration of single-cell and multicellular wound responses. Annu Rev Cell Dev Biol. 2011; 27: 237–263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZakaria ZA, Kumar GH, Mat Jais AM, et al.: Antinociceptive, antiinflammatory and antipyretic properties of Channa striatus fillet aqueous and lipid-based extracts in rats. Methods Find Exp Clin Pharmacol. 2008; 30(5): 355–362. PubMed Abstract\n\nZakaria ZA, Mat Jais AM, Goh YM, et al.: Amino acid and fatty acid composition of an aqueous extract of Channa striatus (Haruan) that exhibits antinociceptive activity. Clin Exp Pharmacol Physiol. 2007; 34(3): 198–204. PubMed Abstract \| Publisher Full Text\n\nFarouk Musa A, Soni T, Dillion J, et al.: Dataset 1 in: A Study on the Effect of Haruan Fish Extract (Channa striatus) on Wound Healing and Quality of Life of Coronary Artery Bypass Grafting (CABG) Patients: A Prospective, Double-Blind, Randomized, Controlled Trial. F1000Research. 2018. Data Source\n\nFarouk Musa A, Soni T, Dillion J, et al.: Dataset 2 in: A Study on the Effect of Haruan Fish Extract (Channa striatus) on Wound Healing and Quality of Life of Coronary Artery Bypass Grafting (CABG) Patients: A Prospective, Double-Blind, Randomized, Controlled Trial. F1000Research. 2018. Data Source" }	[ { "id": "44565", "date": "30 Apr 2019", "name": "Mahmood Moshiri", "expertise": [ "Reviewer Expertise Pathologist", "Cardiovascular Pathology Experience (10 years)", "Clinical Nutritionist (FICN)." ], "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 think this is an excellent paper and is adding more knowledge to the literature. I really like the conceptualization of assessing the effect of Haruan fish extract (Channa striatus) on chest and leg wounds post-coronary artery bypass grafting (CABG) surgery. Dr Musa and his co-authors have discussed appropriately in the context of the current literature. Interestingly, the authors have used suitable methods and in my opinion, they have provided sufficient information and source data to allow other researchers to repeat different steps of wound healings in other organs of the body. I believe, no additional changes are required. The design, including sample size, wound infection, mobilization, health related quality of life, production of Haruan capsules, specifically the flow chart, and the methods, are perfectly prepared. Results are presented accurately and the conclusions are justified and supported by the data.\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": "61255", "date": "23 Mar 2020", "name": "Brian Olshansky", "expertise": [ "Reviewer Expertise Cardiology", "electrophysiology", "alternative medical therapies" ], "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 reviewer congratulates the investigators for undertaking a challenging study that considers wound healing in patients undergoing coronary artery bypass graft surgery. The authors delineate the methodology for performing this double-blind randomized placebo-controlled trial that compares use of the Haruan fish extract to a placebo to evaluate wound healing and quality-of-life in patients undergoing coronary artery bypass graft surgery. The authors state that wound healing is a primary problem in surgical cases and, based on preliminary information, they study the Haruan fish extract. It would be good to have a standardized extract to test and to determine which may be the active components of this extract. In this study, the design seems reasonable and the outcome measures of wound pain mobilization and health-related quality-of-life using the Nottingham Health Profile is appropriate. In the abstract, the authors state that those who received the Haruan capsule showed better wound healing and had better pain scores as well as improvement in quality-of-life. The analyses seem appropriate as does the determination of the size of the study. Unfortunately only 72.3% completed the study in up to three months of follow-up. Additionally, the study applies mainly to males of the Malay background. Unfortunately, in the results section, there is very little evidence that the Haruan capsule actually had any benefit. It would be good to have some tables and charts demonstrating the efficacy, or not, with Haruan capsule. With regard to mean hospital stay, the results were not significant. Perhaps the study was underpowered to determine significance but, nevertheless, the results were not significant. In terms of the ASEPSIS score, there were no significant differences throughout the follow-up. Additionally, there were no significant differences in mean pain score using the visual analog scale and no difference in mobilization. With regard to the health related quality-of-life, there were no significant differences at discharge and in follow-up. Therefore, this reviewer is puzzled that the abstract states benefit when the results do not show this. Much of the issue may be related to limitations but this manuscript does not confer evidence that the Haruan capsule actually has benefit. Further, any hypothesis that wound healing would be improved by the Haruan extract is not supported here. In the discussion there is enthusiasm about using this Haruan extract with statements that this extract actually improves wound healing but it did not. This reviewer does not understand how the authors can implicate benefit or even discuss mechanisms when there is no evidence based on the results that this actually works. One other thing relates to the costs of hospitalization mentioned in the conclusion but not discussed in any detail 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? 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? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/7-469
https://f1000research.com/articles/7-49/v1	12 Jan 18	{ "type": "Case Report", "title": "Case Report: Polyarteritis nodosa or complicated Henoch-Schonlein purpura, a rare case", "authors": [ "Sajad Hasanzadeh", "Seyedeh Maryam Alavi", "Elahe Masnavi", "Maryam Rohani", "Saeid Jokar", "Sajad Hasanzadeh", "Seyedeh Maryam Alavi", "Elahe Masnavi", "Maryam Rohani" ], "abstract": "Background: Polyarteritis nodosa (PAN) is a vasculitis that affects medium sized arteries. PAN is a rare disease and requires a high vilgilance for diagnosis. For instance, PAN and Henoch-Schonlein purpura (HSP) have narrowing differential diagnosis. Here, we report a case of PAN. Case presentation: Our patient was a 65 year old woman that came to hospital due to abdominal pain and skin lesion on the right upper and right lower extremities. All rheumatologic tests were negative. A biopsy of the skin lesion was reported as mild hyperkeratosis, slight spongiosis with intact basal layer. The dermis showed moderate to severe perivascular PMN infiltration with vessel wall degeneration and extravasation of RBCs. A colonoscopy reported diffuse mucosal erythema and erosions were seen in the rectum until 6cm of anal verge. An electromyogram test and nerve conduction velocity study of the upper extremities reported bilateral mild carpal tunnel syndrome, and in the right lower extremities mononeuritis multiplex could not be ruled out. Abdominopelvic CT scan reported diffuse wall thickening of terminal ileum associated with mesenteric fat and narrow enhancement of inferior Mesenteric artery with patchy filling defect. After evaluation, the patient received corticosteroid pulses plus cyclophosphamide. Conclusion: Diagnosis and treatment of PAN is important and PAN should be considered in a patient with skin lesions and neurological impairment.", "keywords": [ "Polyarteritis nodosa", "Henoch-Schonlein purpura", "vasculitis" ], "content": "Introduction\n\nPolyarteritis nodosa (PAN) is a systemic vasculitis that mostly involves medium sized arteries, and sometimes involves small arteries1. The prevalence of PAN is estimated to be 2 to 33 million individuals worldwide2,3. The annual incidence in some areas of Europe estimate 4.4 to 9.7 per million population4. The diagnosis is most commonly made in middle-aged or older adults, and increases with age, and its peak is in the sixth decade of life2. Polyarteritis nodosa can mimic the clinical manifestations of Henoch-Schonlein purpura (HSP). It is difficult to differentiate between PAN and HSP at an early stage. If PAN is not diagnosed and treated at an early stage it has a high morbidity5. Considering that PAN is a rare disease and requires a high clinical suspicion for diagnosis, here, we report a case of PAN and the reasoning behind its diagnosis in our patient.\n\n\nCase report\n\nThe patient was a 65 year old woman from the south of Iran that came to our hospital due to abdominal pain and skin lesion on right upper and right lower extremities, which were was mostly on the distal of extremities since 2 weeks preadmission. Other complaints of the patient were diarrhea, vomiting, chills, fever and anorexia. In the past medical history, the patient had diabetes, hypertension and Bell's palsy (treated with 40mg prednisolone daily).\n\nOn examination of the skin, the patient had palpable plaque in the erythematous and purpuric context with vesicular and bulla lesion on right upper and right lower extremities that mostly extended to the distal part (Figure 1). An abdominal examination revealed mild tenderness in the epigaster. The extremities were warm and end pulses were normal. Neurologic exam of the right lower extremity revealed decreased motor function (muscle power 4/5).\n\nLaboratory tests: HCV, HBV, HIV, ANA (antinuclear antibodies), crayoglobulin, anti-double-stranded DNA (dsDNA) antibodies, complement (C3 and C4), perinuclear antineutrophil cytoplasmic antibodies (P-ANCA and C-ANCA), all were normal. Urine analysis, amylase and lipase levels were normal. ESR was 40mm/h (normal range, <20mm/h), occult blood one pluses positive, and hemoglobin was 11/9 g/L (normal range, 13–16g/l).\n\nSkin biopsy: Mild hyperkeratosis, slight spongiosis with intact basal layer. The dermis showed moderate to severe perivascular PMN infiltration with vessel wall degeneration and extravasation of RBCs. A diagnosis of a vasculitis leukocytoclastic variant (immunofluorescence is not available at our center).\n\nEvaluation of patient anemia and GI tract were done via endoscopy and colonoscopy.\n\nEndoscopy: Patchy erythematous lesions were observed.\n\nAbdominopelvic CT scan (Figure 2): A 130mm of segment of terminal ileum had diffuse wall thickening (3–8mm) associated with mesenteric fat. Narrow enhancement of inferior mesenteric artery with patchy filling defect, poor enhancement of terminal branches. Therefore, suspicions were: 1)vasculitis, 2)mesenteric ischemia.\n\nNarrow enhancement of the inferior mesenteric artery can be observed (blue arrow).\n\nColonoscopy: Diffuse mucosal erythema and erosions were seen in the rectum until 6cm of anal verge. Hemorrhoid without active bleeding in anus, few erythema and ophtus ulcer in cecum. Terminal ileum was not intubated. A diagnosis of a rectal erosion maybe due to vasculitis.\n\nElectromyogram test and nerve conduction velocity: Upper extremities reported bilateral mild carpal tunnel syndrome, and in right lower extremities mononeuritis multiplex could not be ruled out.\n\nEchocardiography: No evidence of any other disorder.\n\nFinal diagnosis: Vasculitis (PAN or complicated HSP)\n\nThe patient received 1000 mg methylprednisolone IV pulse daily for 3 days, and 750mg cyclophosphamide IV pulse every two weeks for 3 weeks.\n\nAfter 24 hours of receiving treatment, the symptoms of the patient subsided, and after one week improved skin lesions. Currently, the patient is being treated with 50mg prednisolone daily and then we will taper this amount.\n\n\nDiscussion\n\nUnlike other vasculitis, such as microscopic polyarthritis or Wegener’s, PAN is not associated with ANCA6. The organs most affected in PAN are the skin, renal and GI tract. Cardiac involvement can manifest itself with hypertension, or even ischemic heart disease7. In the skin, PAN may manifest by erythematous nodules, livedo reticularis, ulcer, bullous or vesicular eruption and purpura6,8,9. Gastrointestinal symptoms that may be seen include abdominal pain, nausea, vomiting, melena, and bloody or non-bloody diarrhea10. One of the most common manifestations of patients with PAN is mononeuropathy multiplex that typically involves both motor and sensory deficits in up 70% of patients6,11. Most cases of PAN are idiopathic, although hepatitis B virus infection, hepatitis C virus infection, and hairy cell leukemia are important in the pathogenesis of some cases3,4,12,13. PAN can mimic the clinical manifestations of HSP. It is difficult to differentiate between PAN and HSP at an early stage5. The biopsy pattern helps to differentiate between PAN and HSP; in tissue studies of HSP leukocytoclastic vasculitis in post capillary venules together with IgA deposition is observed14. As already mentioned, PAN is most commonly seen in middle-aged or older adults3, while HSP is a childhood disease that occurs between the ages of 3 and 15 years15. Neurologic manifestation in HSP is rare. Single reports and case series document neurologic manifestations including headaches, intracerebral hemorrhage, focal neurologic deficits, ataxia, seizures, and central and peripheral neuropathy in children with HSP16. In the present case, using clinical manifestations and laboratory tests, we excluded other differential diagnosis apart from PAN. Considering that PAN and HSP have narrowing clinical manifestation, we differentiated between the two diseases by age and neuropathy. However, although the diagnosis of the present patient is PAN, for a better diagnosis, immunofluorescence of the biopsy is needed, which is not available in our center. Overall, diagnosis and treatment of PAN is important, and PAN should be considered in a patient with skin lesions and neurological impairment.\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 author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSato O, Cohn DL: Polyarteritis and microscopic polyangiitis. In: Rheumatology, Klippel JH, Dieppe PA (Eds.), Mosby, St Louis; 2003.\n\nMahr A, Guillevin L, Poissonnet M, et al.: Prevalences of polyarteritis nodosa, microscopic polyangiitis, Wegener's granulomatosis, and Churg-Strauss syndrome in a French urban multiethnic population in 2000: a capture-recapture estimate. Arthritis Rheum. 2004; 51(1): 92–9. PubMed Abstract \| Publisher Full Text\n\nRamos-Casals M, Muñoz S, Medina F, et al.: Systemic autoimmune diseases in patients with hepatitis C virus infection: characterization of 1020 cases (The HISPAMEC Registry). J Rheumatol. 2009; 36(7): 1442–8. PubMed Abstract \| Publisher Full Text\n\nGuillevin L, Mahr A, Callard P, et al.: Hepatitis B virus-associated polyarteritis nodosa: clinical characteristics, outcome, and impact of treatment in 115 patients. Medicine (Baltimore). 2005; 84(5): 313–22. PubMed Abstract \| Publisher Full Text\n\nOzen S, Anton J, Arisoy N, et al.: Juvenile polyarteritis: results of a multicenter survey of 110 children. J Pediatr. 2004; 145(4): 517–22. PubMed Abstract \| Publisher Full Text\n\nKallenberg CG, Brouwer E, Weening JJ, et al.: Anti-neutrophil cytoplasmic antibodies: current diagnostic and pathophysiological potential. Kidney Int. 1994; 46(1): 1–15. PubMed Abstract \| Publisher Full Text\n\nPagnoux C, Seror R, Henegar C, et al.: Clinical features and outcomes in 348 patients with polyarteritis nodosa: a systematic retrospective study of patients diagnosed between 1963 and 2005 and entered into the French Vasculitis Study Group Database. Arthritis Rheum. 2010; 62(2): 616–26. PubMed Abstract \| Publisher Full Text\n\nGibson LE, Su WP: Cutaneous vasculitis. Rheum Dis Clin North Am. 1995; 21(4): 1097–113. PubMed Abstract\n\nKarlsberg PL, Lee WM, Casey DL, et al.: Cutaneous vasculitis and rheumatoid factor positivity as presenting signs of hepatitis C virus-induced mixed cryoglobulinemia. Arch Dermatol. 1995; 131(10): 1119–23. PubMed Abstract \| Publisher Full Text\n\nLevine SM, Hellmann DB, Stone JH: Gastrointestinal involvement in polyarteritis nodosa (1986–2000): presentation and outcomes in 24 patients. Am J Med. 2002; 112(5): 386–91. PubMed Abstract \| Publisher Full Text\n\nMoore PM: Neurological manifestation of vasculitis: update on immunopathogenic mechanisms and clinical features. Ann Neurol. 1995; 37 Suppl 1: S131–41. PubMed Abstract \| Publisher Full Text\n\nHasler P, Kistler H, Gerber H: Vasculitides in hairy cell leukemia. Semin Arthritis Rheum. 1995; 25(2): 134–42. PubMed Abstract \| Publisher Full Text\n\nCarpenter MT, West SG: Polyarteritis nodosa in hairy cell leukemia: treatment with interferon-alpha. J Rheumatol. 1994; 21(6): 1150–2. PubMed Abstract\n\nJennette JC, Falk RJ: Small-vessel vasculitis. N Engl J Med. 1997; 337(21): 1512–23. PubMed Abstract \| Publisher Full Text\n\nGardner-Medwin JM, Dolezalova P, Cummins C, et al.: Incidence of Henoch-Schönlein purpura, Kawasaki disease, and rare vasculitides in children of different ethnic origins. Lancet. 2002; 360(9341): 1197–202. PubMed Abstract \| Publisher Full Text\n\nNadrous HF, Yu AC, Specks U, et al.: Pulmonary involvement in Henoch-Schönlein purpura. Mayo Clin Proc. 2004; 79(9): 1151–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "29766", "date": "25 Jan 2018", "name": "Marco de Vincentiis", "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 case of a 65 year old woman that was admitted to the authors' hospital due to abdominal pain and skin lesion on the right upper and right lower extremities, with negative rheumatologic tests. After careful diagnostic workout, the final diagnosis was vasculitis (Polyarteritis nodosa or complicated Henoch-Schonlein purpura). The paper is interesting, also due to the rarity of the disease. However, there are some points that need to be improved before final approval:\nThe authors report Bell's Palsy in patient's medical history; it would be interesting to know when this occurred. Since this was treated with corticosteroid therapy, it is important to know if it was present when the patient already had PAN lesions - and in this case if they also improved during therapy - or if it occurred prior to PAN-related lesions arose.\n\nTiming details are missing: in the case presentation - follow up and outcomes - the authors report that the patient \"is being treated with 50mg prednisolone daily and then we will taper this amount\". It is important to define for how long the patient is being treated, how much time past from initial diagnosis and therapy, and how the authors intend to reduce treatment.\n\nSensorineural Hearing Loss is often reported in PAN and, in some cases, may occur as the presenting symptom. Hearing Loss is typically bilateral and symmetrical, with sudden or rapidly progressive onset. It would be interesting to know if hearing impairment was reported by the patient, or investigated.\n\nIn the discussion we recommend to further discuss the findings that led to PAN diagnosis in this case.\n\nThe paper has several English language typos and grammar mistakes that should be corrected by a native English speaker (i.e. \"which were was mostly\", etc).\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? Yes", "responses": [ { "c_id": "3387", "date": "30 Jan 2018", "name": "Saeid Jokar", "role": "Author Response", "response": "Hi Marco,Thank you for attention in review of our article. I will correct the article by your statements. One week pre-admission the patient was under corticosteroid treatment because bell's palsy. After 2 weeks, if the no recurrent of patient symptoms we will taper off corticosteroids amount 10 %. We will reduce the dose of corticosteroids until reaches to control of patient symptoms, then by the patient condition will decide about it. Thank you - I add your statement to the manuscript Other comments are considered." } ] }, { "id": "31810", "date": "13 Mar 2018", "name": "Patricia Woo", "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 indeed a severe case of systemic vasculitis. The criteria used to classify the combined clinical and histological findings are not clearly referenced for discussion. The EULAR/PRINTO/PRES criteria published in 2016 are more discriminatory with the addition of IgA immunofluorescence for IgA associated vasculitis (formerly known as HSP). Unfortunately the authors were not able to perform this test and the skin histology did not describe a necrotizing vasculitis. The authors are correct that mononeuritis multiplex would be a separator, but the evidence in this case is more circumstantial as reported. The time line of Bells palsy is suggestive. The nerve conduction report is equivocal. Renal dysfunction or joint involvement were not mentioned.\n\nBoth conditions can respond well to prednisolone alone, or in combination with cyclophosphamide depending on the severity.\n\nSince the case report is to highlight the overlapping features of the vasculitides, I agree that the differentiating diagnostic features are important.\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? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "3509", "date": "16 Apr 2018", "name": "Saeid Jokar", "role": "Author Response", "response": "Hi Patricia I corrected the article in accordance with your comments. Thank you for advising us on improving the content of the article. Best Regards" } ] } ]	1	https://f1000research.com/articles/7-49
https://f1000research.com/articles/6-2162/v1	21 Dec 17	{ "type": "Software Tool Article", "title": "RSEQREP: RNA-Seq Reports, an open-source cloud-enabled framework for reproducible RNA-Seq data processing, analysis, and result reporting", "authors": [ "Travis L. Jensen", "Michael Frasketi", "Kevin Conway", "Leigh Villarroel", "Heather Hill", "Konstantinos Krampis", "Johannes B. Goll", "Travis L. Jensen", "Michael Frasketi", "Kevin Conway", "Leigh Villarroel", "Heather Hill", "Konstantinos Krampis" ], "abstract": "RNA-Seq is increasingly being used to measure human RNA expression on a genome-wide scale. Expression profiles can be interrogated to identify and functionally characterize treatment-responsive genes. Ultimately, such controlled studies promise to reveal insights into molecular mechanisms of treatment effects, identify biomarkers, and realize personalized medicine. RNA-Seq Reports (RSEQREP) is a new open-source cloud-enabled framework that allows users to execute start-to-end gene-level RNA-Seq analysis on a preconfigured RSEQREP Amazon Virtual Machine Image (AMI) hosted by AWS or on their own Ubuntu Linux machine. The framework works with unstranded, stranded, and paired-end sequence FASTQ files stored locally, on Amazon Simple Storage Service (S3), or at the Sequence Read Archive (SRA). RSEQREP automatically executes a series of customizable steps including reference alignment, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially expressed genes, heatmaps, co-expressed gene clusters, enriched pathways, and a series of custom visualizations. The framework outputs a file collection that includes a dynamically generated PDF report using R, knitr, and LaTeX, as well as publication-ready table and figure files. A user-friendly configuration file handles sample metadata entry, processing, analysis, and reporting options. The configuration supports time series RNA-Seq experimental designs with at least one pre- and one post-treatment sample for each subject, as well as multiple treatment groups and specimen types. All RSEQREP analyses components are built using open-source R code and R/Bioconductor packages allowing for further customization. As a use case, we provide RSEQREP results for a trivalent influenza vaccine (TIV) RNA-Seq study that collected 1 pre-TIV and 10 post-TIV vaccination samples (days 1-10) for 5 subjects and two specimen types (peripheral blood mononuclear cells and B-cells).", "keywords": [ "RSEQREP", "RNA-Seq", "transcriptomics", "differential gene expression", "pathway enrichment", "reproducible research", "cloud computing", "trivalent influenza vaccine" ], "content": "Introduction\n\nThe advent of next-generation sequencing (NGS) technologies has dramatically reduced costs and thus democratized sequencing1. Consequently, both big research consortia and small laboratories now have the ability to utilize large-scale genomic applications such as RNA sequencing (RNA-Seq) for transcriptome profiling. However, while sequencing cost is on the decline, the cost of data storage, analysis and interpretation is increasing1. Major challenges for analyses of RNA-Seq data include the need for a substantial informatics hardware and software infrastructure as well as a wide range of computational skills to effectively manage and process the data. With the plethora of published bioinformatics software, data formats, and human genome information, careful bioinformatics workflow development, parameterization, reference dataset management, and execution are required to generate consistent, reproducible and high-quality analysis datasets2. Interpretation of RNA-Seq data requires special statistical and visualization techniques3,4. In addition, most of the NGS bioinformatics software only runs on the Linux operating system (OS) or is dependent on Linux tools/utilities. These requirements limit the ability of small labs and individual principal investigators to analyze such data, in particular, those that use desktop computers running non-Linux based OS with limited IT support. Emerging information technologies, bioinformatics workflow engines, and open-source analytical modules are presenting opportunities to reduce this burden5. Virtualization technologies, for example, now allow entire OS replete with all the necessary software packages to be archived and then instantiated just about anywhere at a moment’s notice, independent of the hardware architecture available. For instance, all software components and dependencies can be encapsulated within Virtual Machines (VMs). These characteristics of virtual appliances allow users to choose the number and size of VMs to be provisioned and thus provide on-demand computational scalability. Commercial cloud service providers such as Amazon Web Services, Google Cloud Platform, and Windows Azure provide user-friendly web-based tools to manage VMs and associated computational resources, including cloud storage, networking, security, identity management, and backup and disaster recovery. This pay-as-you go model eliminates upfront capital expenses by converting the budgeting representation of bioinformatics processing tasks and storage into well-defined operational costs. The open-source R statistical programming language in combination with the Bioconductor package resource provides researchers with a consistent way to share and use specialized statistical methods for RNA-Seq analysis6,7. In combination with the R knitr package, analysis data sets can be processed automatically using R and summarized in reports by integrating formatting instructions with analytical components8. Together, these technologies can reduce analysis time and programming effort, allow more accurate estimation of hardware costs, improve quality of results, and facilitate reproducible research by transparently documenting all steps including software and OS.\n\nRNA-Seq allows snapshot measurements of the human transcriptome by partially sequencing reverse-transcribed RNA transcripts (cDNA) expressed in cell populations or single cells of interest. In the context of clinical trials, the goal of transcriptomics studies is to identify and better understand changes in cell states on the gene expression level that can be attributed to a certain treatment (e.g., a vaccine or drug)9,10, or changes that can predict individual treatment responses (e.g. the likelihood of developing protective levels of antibody)11,12. The number of RNA-Seq reads (short DNA sequence) corresponding to a transcript has been shown to be linearly associated with true transcript abundance spanning a large quantitative range13. Prior to gene expression quantification, processing of human RNA-Seq data requires a computationally intensive alignment step that maps sequence reads against the human reference transcriptome and/or genome sequence14–16. Resulting alignment metrics including genomic mapping locations (chromosome and position), alignment information (insertions, deletions, and matching bases), alignment quality scores, among other information, are recorded in the form of Binary Alignment Mapping (BAM) files17. Various algorithms have been developed that use this mapping information for determining/counting which sequence read originated from a certain gene, gene isoform, or gene exon18–23. Following gene expression quantification, key analysis steps include the detection of treatment-responsive genes (e.g. 4) and subsequent characterization of these genes using pathway enrichment analysis (e.g. 24). Challenges prior to RNA-Seq data interpretation include (1) estimation of expected cost for storage and data processing, (2) provisioning of computational resources for storage and data processing, (3) installation of Linux OS, required bioinformatics software, and reference data sets, (4) suitable analytical methods including advanced data visualizations to summarize key tends in the data, and (5) automation and documentation of all steps to facilitate reproducible research.\n\nIn this article, we summarize the RSEQREP framework we developed that allows researchers to address these challenges and to streamline the transition from a desktop environment to a server-based scalable cloud infrastructure using Amazon Web Services (AWS). Alternatively, the framework can be installed on a local Ubuntu machine via installation scripts that we provide. We exemplify the framework’s capabilities using RNA-Seq data generated for an influenza vaccine study that extracted RNA from peripheral blood mononuclear cells (PBMCs) and B-cells samples collected from 5 subjects prior to trivalent influenza vaccine (TIV) vaccination and at 10 time points post TIV vaccination (days 1-10) (GEO accession: GSE45764, Dataset 1,10).\n\n\nMethods\n\nFigure 1 provides an overview of RSEQREP software components. The framework is organized into four main components: (1) reference data setup, (2) pre-processing, (3) analysis, and (4) reporting. The pre-processing component uses a combination of open-source software, shell, R, and Perl scripts and a SQLite relational database to process raw sequence data, quantify gene expression, and track storage, file check-sums, CPU, memory, and other runtime metrics. The analysis component is based on R using both custom R programs, as well as existing R/Bioconductor packages. The reporting component is based on R, the knitr R package, and LaTeX for reproducible and automatic PDF report and figure/table generation. All components read user-defined arguments from the respective tab in the RSEQREP configuration spreadsheet (RSEQREP/config/config.xls).\n\nRSEQREP provides a reproducible start-to-end analysis solution for RNA-Seq data by automating (1) reference dataset initialization/download, (2) RNA-Seq data processing (3) RNA-Seq analysis, and (4) reporting including a summary PDF report and publication-ready table and figure files. Steps can be run in a modular fashion and key computational metrics are tracked in a SQLite database. The software runs on a pre-configured RSEQREP AMI or on a local Ubuntu Linux machine. Users can customize individual steps and enter their experimental design information via an Excel configuration file.\n\nAll four workflow components can be run in sequence via the RSEQREP/run-all.sh script or run individually to update results of the respective component. When running each individual step, the most recent version of the configuration file will be reloaded to ensure that any modifications to the configuration will be reflected. This is particularly useful for optimizing results and customizing result presentation, for example, by removing outliers, optimizing the low-expression cut-off, or adjusting the color-coding range for heatmaps. In the following, we provide an overview of each of these steps. Additional information can be found in the method section of the RSEQREP summary report (Supplementary File S1).\n\nStep 1) Reference Data Set-up. The RSEQREP/setup.sh script reads all user-specified arguments provided in the config.xls file, downloads all required reference data including user-specified versions of the human reference genome sequence and associated gene model information from the Ensembl database25. Input for pathway enrichment analysis is handled via Gene Matrix Transposed (GMT) files. For GMT files, Entrez Gene IDs, Ensembl Gene IDs, or gene symbols are supported and will be automatically mapped to the human Ensembl reference annotations. We recommend that users obtain reference pathway GMT files from the Molecular Signatures Database (MSigDB)26. The MSigDB import is not automated as download requires registration but the location of downloaded GMT file can be specified in the configuration file. We do provide a script (RSEQREP/source/shell/download-gene-sets.sh) to automatically download Reactome, Blood Transcription Module27, and KEGG pathway information and convert this information to GMT files (note, a license may be required prior to downloading KEGG pathway information). Following the reference dataset download, an index of the human reference genome sequence will be created to optimize reference alignment searches15,16. Result files generated as part of this step are saved under the data output directory.\n\nStep 2) Data Pre-processing. Based on FASTQ file input specifications in the config.xls, the RSEQREP/run-pre-processing.sh script downloads and decrypts (optional) FASTQ files hosted on AWS Simple Storage Service (S3) storage (https://aws.amazon.com/s3), a local file location (Linux file path), or directly from Sequence Read Archive (SRA)29 via FTP URLs. Following the download, the script executes sequence data QC (FastQC), reference genome alignments (STAR16 or Hisat215 splice-aware aligner on stranded, unstranded, or paired-end read data as specified in the config.xls), reference based compression to generate storage-optimized CRAM files (SAMtools17), gene expression quantification (featureCounts as implemented in subread18), and reference genome alignment QC (RSeQC30). Additionally, the script tracks program arguments, program return codes, input and output file names, file sizes, MDS checksums, wall clock times, CPU times and memory consumption in a SQLite relational database. Interim result files generated as part of this step are saved under the specified pre-processing output directory.\n\nStep 3) Data Analysis. The RSEQREP/run-analysis.sh script initializes analysis datasets for the final reporting step including (1) TMM-normalization31 and exclusion of low-expressed genes, (2) principal component analysis (PCA), distance matrix calculations for non-metric multidimensional scaling (MDS), and hierarchical clustering for global multivariate analyses, (3) log2 fold change calculations used as input for heatmap and co-expressed gene-cluster analyses, (4) identification of differentially expressed (DE) genes (edgeR32), co-expressed gene clusters (pvclust33), and enriched pathways (GoSeq24). Interim result files generated as part of this step are saved under the specified report output directory.\n\nStep 4) Automatic Report Generation. The RSEQREP/run-report.sh script produces the final results. It runs R analyses on the intermediate analysis files generated in Step 3, generates a summary PDF report using the knitr R package in combination with LaTeX, and result tables in gzipped .csv format as well as individual figure files in .pdf, and .png format. This script also summarizes key run time statistics that were collected as part of Step 2. Result files generated as part of this step are saved under the specified report output directory.\n\nA 35 GiB Elastic Block Store (EBS) volume, i.e. storage immediately accessible to the OS (http://docs.aws.amazon.com/AWSEC2/latest/UserGuide/EBSVolumes.html), sufficiently covers space for the OS, user accounts, reference data, and to process and analyze dataset sizes similar to that of the influenza vaccine case study when CRAM compression is deactivated. To accommodate storage for CRAM-compressed files and studies with larger sample sizes and/or sequence coverage, additional EBS volumes are required (see information on AWS set-up under RSEQREP/aws/aws-setup.docx).\n\nWe found that a c3.xlarge computational Elastic Compute Cloud (EC2) instance type (4 vCPUs, 7.5 GiB, https://aws.amazon.com/ec2/instance-types) is sufficient for data processing and analysis, but a higher memory machine (c3.4xlarge: 16 Gib for HISAT2 and c3.8xlarge: 37 Gib for STAR) is required to successfully complete the indexing of the reference genome sequence as part of Step 1.\n\nWe provide a pre-configured RSEQREP Amazon Virtual Machine Image available on AWS at (https://aws.amazon.com, AMI ID: RSEQREP (RNA-Seq Reports) v1.0 (ami-e1708b9b)) that combines the Ubuntu operating system Version 16.04.2 (long-term support) with all additional software that is required for RSEQREP operation (RSEQREP/SOFTWARE.xlsx). We prepared a manual that provides instructions on how to set-up an AWS instance including mounting of EBS volumes for local storage and an optional Elastic IP address for machine access (RSEQREP/aws/aws_instructions.docx). Alternatively, we provide installation scripts that can be executed on a local Ubuntu machine (Version 16.04.2) to install necessary dependencies (RSEQREP/ubuntu/install-software.sh). In both cases, AWS and local set-up, prior to workflow execution, users would need to pull the latest RSEQREP source code from GitHub (git clone https://github.com/emmesgit/RSEQREP).\n\nRSEQREP configuration is handled via the RSEQREP/config/config.xlsx file. The first tab allows users to specify sample metadata. Fields include subject ID, sample ID, sampling time point, a flag (is_baseline) that indicates if a sample was collected prior to treatment, the treatment group, specimen type (e.g. B-cells, PBMCs, etc.), and FASTQ sequence file location (AWS S3, local, or remote SRA location). In addition, color-coding for time points, treatment groups, and specimen types can be defined. The second tab specifies options related to the pre-processing step. This tab uses a two-column key value pair format to define options. For example, to specify the Ensembl database version 74, users can set the version value via the ensembl_version key value pair to 74. Other options include the type of RNA-Seq data (stranded: yes/no) and reference alignment software (Star or Hisat2). Paired-end experiments can be accommodated for each sample by specifying two input FASTQ files. The third tab allows users to customize analysis and reporting components. Options include specification of cut-offs to define lowly-expressed genes, DE genes, and enriched pathways, as well as the distance metric and hierarchical clustering algorithm used for heatmap and gene clustering analysis. For further information, see descriptions and examples for each of these options in the influenza vaccine case study configuration file (Supplementary File S2). We implemented the framework to dynamically adjust the report presentation depending on the number of subjects, time points, specimen types, and treatment group combinations. For example, Venn diagrams are shown for comparisons between up to five sets (e.g. five time points). Larger sets are accommodated via UpSet plots34. The configuration file allows users to carry out subgroup analysis by limiting the metadata file to samples, treatment groups, and time points of interest.\n\n\nUse Case\n\nTo illustrate the capabilities of RSEQREP, we analyzed a publicly available RNA-Seq dataset comprising 110 RNA-Seq samples: five subjects, 11 time points (pre-vaccination and days 1-10 post-vaccination), two specimen types (PBMCs and B-cells), and one treatment group (Trivalent Influenza Vaccination (TIV)) (GEO accession: GSE45764, Dataset 1,10). The unstranded single-end RNA-Seq experiment was carried out with a read length of 65 nt (nucleotides) and an average sequence coverage of 16 million total mapped reads. The study was designed to obtain detailed information on the early temporal gene expression response following TIV vaccination in both PBMC and B-cells. The configuration file that specifies the case study experimental design, public SRA FASTQ FTP URLs, data processing and analysis parameters is provided in Supplementary File S2. The configuration file allows users to reproduce RSEQREP results for this case study on their own RSEQREP AWS instance or Ubuntu Linux machine. Supplementary File S1 represents the corresponding RSEQREP Summary PDF report, including 131 figures and 135 tables. In the following, we describe a subset of key findings (referenced supplemental tables and figures refer to the corresponding results in the supplemental PDF report). See Supplementary File S1 methods for additional information on pre-processing and analysis steps.\n\nPCA results revealed that most variation in gene expression based on standardized log2 counts per million across all 110 samples was attributable to cell type (B-cells vs. PBMCs, Figure 2). In addition, two extreme outliers, including one B-cell sample that was likely mislabel as a PBMC sample, were identified. These samples were added to the configuration file as outliers to be excluded from downstream analysis. Negative binomial models as implemented in the edgeR package32 were fit to identify genes that were DE compared to pre-vaccination at each of the post-vaccination days. UpSet plots visualizing the number and overlap of DE genes over time are presented in Figure 3. PBMCs showed overall peak DE responses at day 1 (24 hours after TIV vaccination) with 136 genes being DE compared to pre-treatment gene expression levels. Between days 1–4, PBMC DE signals declined followed by a broader second peak response for days 5–8 reaching the second highest response of 96 DE genes at day 6. While most DE genes in PBMCs at day 1 were unique (106 of 136 genes (78%)), most DE genes at day 6 (64 of 96 (67%)) were overlapping with other DE gene responses, in particular, with days 5, 7, and 8. In contrast to PBMCs, B-cells did not exhibit a noticeable DE gene signal at day 1, but showed responses between days 5–8 (121–481 genes) reaching highest responses at day 6 (481 genes). While some DE genes were unique to day 6 (168 of 481 (35%)), many were shared with day 7 (124 genes), as well as day 7 and day 8 (72 genes). For both cell types, most DE genes were up-regulated from pre-vaccination (Figure 3, middle panel vs. right panel). Most of the overlap between PBMC and B-cell DE genes was observed at day 6, at which 62 of 96 DE PBMC genes (65%) were also reported as DE in B-cells (Figure S35). Tables S7–S26 list individual DE gene results. In the following, pathway enrichment analysis results for peak DE responses and a selection of identified co-expressed gene clusters are summarized.\n\nRSEQREP supports multivariate visualizations, including principal component analysis (PCA) to visualize key trends in the data. The analysis uses standardized log2 counts per million (mapped reads) for genes that met the low expression cut off as input. As shown for the influenza case study, the PCA analysis indicated that PBMC (highlighted in red) and B-cell (highlighted in blue) samples differ substantially in their transcriptional profiles. In addition, two outliers were identified in relation to the other samples (highlighted in blue circles). Ellipses represent the 95% confidence interval for the bivariate mean based on the first two principal components by specimen type.\n\nThese panels summarize the DE gene overlap between post-treatment days for up- or down-regulated DE genes (shown to the right in black), for up-regulated DE genes (shown in the middle in red), and down-regulated DE genes (shown to the right in blue), respectively within specimen type (B-cells are shown in the top row, PBMCS in the bottom row). In each panel, the bottom left horizontal bar graph labeled SDEG Set Size shows the total number of DE genes per post-treatment time point. The circles in each panel’s matrix represent what would be the different Venn diagram sections (unique and overlapping DE genes). Connected circles indicate a certain intersection of DE genes between post-treatment days. The top bar graph in each panel summarizes the number of DE genes for each unique or overlapping combination. In the top left panel, for example, the first vertical bar/column shows those DE genes that are unique to day 6 (168 DE genes). The second shows those DE genes that are shared only between days 6 and 7 (124 DE genes). The third are those DE genes that are shared between days 6, 7, and 8 (72 DE genes), and so forth. As shown for the influenza case study, most of the DE genes for B-cells were detected and overlapped between days 6, 7 and 8 while most of the DE genes for PBMCs were uniquely identified at day 1.\n\nTo functionally characterize DE gene responses, pathway enrichment analysis as implemented in the GoSeq R package24 was carried out using MSigDB (Version 5.2, Dataset 2) and Blood Transcription Modules (Dataset 3) reference gene sets/pathways. Pathway enrichment analysis of the day 1 peak DE gene signal in PBMCs identified innate immune response signaling pathways including Reactome-based interferon signaling, in particular, interferon gamma signaling and interferon alpha/beta signaling (Figure 4, Table S97). Top enriched GO Biological processes included innate immune response, defense response to virus and response to type I interferon (Table S92). The top Blood Transcription Modules indicated that day 1 PBMC DE genes were most preferentially enriched in monocytes (II) (M11.0) but also enriched in activated dendritic cells (II) (M165), and enriched in neutrophils (I) (M37.1) (Table S91). The day 6 PBMC DE gene signal was related to plasmablast and B-cell Blood Transcription Module signatures including plasma cells, immunoglobulins (M156.1), plasma cells, and B cells, immunoglobulins (M156.0), and enriched in B-cells (II) (M47.1) (Table S115). The day 6 peak DE gene response in B-cells was enriched in several cell cycle-related pathways including Reactome cellcycle mitotic, cellcycle and DNA replication (Figure 4, Table S73). In addition, processes involved in protein-processing such as GO Cellular Component endoplasmic reticulum part and endoplasmic reticulum (Table S69) and GO Biological Process protein complex assembly and intracellular protein transport (Table S68), as well as Reactome metabolism of proteins, post-translational protein modification, and asparagine N-linked glycosylation were identified (Figure 4, Table S73). Enrichment results based on Blood Transcription Modules confirmed enrichment of cell cycle-related modules but also identified several plasma cell-related signatures such as plasma cells surface signature (S3), plasma cells, and B cells, immunoglobulins (M156.0), and plasma cells, immunoglobulins (M156.1) (Table S67). The top most enriched MSigDB Immunological Signature was related to genes that were up-regulated at day 7 following TIV vaccination compared to pre-vaccination in a previous influenza vaccine study by Nakaya et al. (GEO accession: GSE29614, 35) (Table S70). Tables S50–S133 list all pathway enrichment analysis results.\n\nReactome pathways that were enriched in at least two conditions are shown. Cells are color-coded by enrichment score: -1 x log10(FDR-adjusted p-value). Cell values represent the number of DE genes that overlap with a certain pathway. Numbers in brackets indicate enriched pathways, i.e. pathways that met the specified FDR-adjusted p-value cut off. Pathways were clustered based on enrichment score. As shown for the influenza case study, pathways related to cell-cycle as well as protein metabolism were enriched in B-cells at day 6. Both, B-cell and PBMCs showed an enrichment of interferon signaling-related pathways at day 1.\n\nTo identify robust clusters of co-expressed DE genes based on correlation between log2 fold change responses, unsupervised multi-scale bootstrap resampling as implemented in the pvclust R package33 was executed. Several known immuno-globulin genes had robustly correlated log2 fold changes across all post-vaccination days (day 1–10) in B-cells and PBMCs reaching peak mean log2 fold change responses between days 5 and 8 (Figure 5). The immunoglobulin gene cluster highlighted for PBMCs comprised 7 genes (5 immunoglobulin genes: IGHG1, IGHG3, IGHGP, IGKC, IGKV3-11 and 2 genes not encoding for immunoglobulins: MZB1, and TNFRSF17) (Figure 5 bottom right). MZB1 is known to play a role in IgM assembly and secretion while TNFRSF17 is known to regulate humoral immunity including plasma cells. Several known interferon-inducible genes co-expressed in PBMCs (IFIT1, IFIT2, and IFIT3) showed an initial peak in log2 fold change response at day 1, which declined to pre-vaccination levels by day 4, followed by a second higher peak response at day 8 (Figure 5 bottom left). Time trends for all identified gene clusters are shown in Figures S79–S86.\n\nRSEQREP supports unsupervised multiscale bootstrap resampling to identify co-expressed gene clusters based on their log2 fold change pattern over time. A subset of trends is shown for the influenza case study. Several co-expressed immunoglobulin genes reached peak log2 fold changes compared to pre-treatment between day 6 and 7 while a cluster of interferon-induced antiviral (IFIT) genes showed an earlier peak in log2 fold change at day 1 in addition to a peak at day 8 in PBMCs.\n\n\nDiscussion\n\nThere is an increasing trend towards more open and transparent research including increasing demands for sharing of source code, software snapshots as well as enhanced scalability to facilitate processing of increasingly larger datasets. A plethora of open-source software for RNA-Seq data processing and analysis has been developed4,36,37. The strength of the RSEQREP framework is its start-to-end open-source solution that combines operating system, bioinformatics software, reference data set-up, data processing, analysis, advanced data visualizations, and automatic reporting. The resulting RNA-Seq PDF reports can easily be customized, extended, and shared.\n\nRSEQREP supports the reproducible research paradigm via its pre-configured AMI, open-source components, user-friendly configuration file, and functionality to rerun analyses from start-to-end or in parts. Using the default RSEQREP AMI, in addition to on-demand scalable computational resources, has the benefit of integrating the operating system and all software installations as part of analysis snapshots referenced in the report, providing for complete transparency and full reproducibility of all components involved. In addition, the software tracks computational runtime metrics (CPU and memory consumption), which can be used to track and estimate computational cost. Towards that end, we benchmarked the preprocessing step for the influenza vaccine case study data (110 samples) using increasingly powerful but also more expensive AWS EC2 instance types: c3.xlarge (4 vCPUs; 7.5 Gib RAM), c3.2xlarge (8 vCPUs; 15 Gib RAM), c3.4xlarge (16 vCPUs; 30 Gib RAM), and c3.8xlarge (32 vCPUs; 60 Gib RAM). We found that the c3.2xlarge (8 vCPUs; 15 Gib RAM) machine marks the ideal convergence of processing time and cost (Figure 6).\n\nMetrics are based on 110 influenza case study RNA-Seq samples. The following instance types were used: c3.xlarge (4 vCPUs, 7.5 GiB Mem), c3.2xlarge (8 vCPUs, 15 GiB Mem), c3.4xlarge (16 vCPUs, 30 GiB Mem), c3.8xlarge (32 vCPUs, 60 GiB Mem). Median wall clock time is summarized as tracked in the RSEQREP SQLite database. The biggest relative reduction in wall clock time across processes was observed when switching from the 4 vCPU to the 8 vCPU instance type (c3.xlarge vs. c3.2xlarge). Higher core machines (16 and 32 vCPUs) did result in further reduced wall clock time for completing reference alignments (HISAT2) and gene expression quantification (Subread) but the change was not as substantial.\n\nRSEQREP includes a collection of best practice analytical tools that we identified through extensive review of the peer-reviewed literature. This includes TMM-normalization to remove systematic differences between samples31, filtering of lowly expressed genes to improve accuracy of fold change estimates and power of DE detection, application of statistical methods that model read count variability using a discrete negative binomial distribution and share information across genes32, the use of moderated log2 counts per million for multivariate analyses, and adjustment for gene length bias38 as part of pathway enrichment analysis39. In addition, the software provides several unique visualizations, including multivariate starplots for reference alignment QC (Figures S2), co-expressed gene cluster time trends (Figure 5), as well as pathway enrichment heatmaps (Figure 4) and radar plots (Figure S117).\n\nRNA-Seq data processing and analysis is a constantly evolving field and there is no consensus on how to best analyze the data. For example, RSEQREP summarizes gene expression on the gene level - a widely used robust gene expression quantification approach18,19. However, methods that support expression quantification on the gene-isoform level have been developed20–23. Depending on the research question, RNA-Seq analysis may include novel transcript/splice junction discovery, determination of single nucleotide polymorphism (SNPs), detection of RNA-editing events, and fusion genes40. In addition, several other popular DE gene detection algorithms such as DESeq2 exist41. While such additional analysis choices are currently not implemented in RSEQREP, the key advantages of this framework are that users have complete access to the source code to make custom updates to all workflow, analysis, and reporting components. In combination with scalable cloud resources this allows for rapid prototyping of analysis reports.\n\nUsing RSEQREP on published RNA-Seq data of an influenza vaccine study10, we confirmed key transcriptional events in PBMCs and B-cells following TIV vaccination10. Three of five subjects in this study had reported previous influenza vaccinations. A memory response was confirmed by the RSEQREP analysis, which identified an early plasma cell and cell proliferation signature in B-cells with a peak 6 days following vaccination. This signal included cluster responses for several co-expressed immunoglobulin genes as well as an up-regulation of genes preferentially involved in protein assembly, protein transport, ER-related pathways – all of which are at the core of antibody-generating cellular machinery. While not as strong as for B-cells, a peak day 6 plasma cell signature and co-expressed immunoglobulin gene response was also identified in PBMCs. This makes sense as B-cells are included in bulk PBMCs. PBMCs showed a strong up-regulation of an innate immune signaling responses 24 hours post-vaccination, in particular, responses related to interferon signaling. This signaling response was enriched in monocyte, dendritic cell, and neutrophil-specific gene expression signatures indicating that it was driven by the innate immune cell subset within PBMCs. Several co-expressed genes in the IFIT gene family were significantly up-regulated at day 1. These genes are known to be activated following interferon signaling and to exhibit antiviral activity by recognizing and inhibiting viral RNA42,43. This is in agreement with other studies that have shown that IFIT genes are up-regulated 24 hours post-influenza vaccination12,44.\n\n\nData and software availability\n\nRSEQREP source code available from: https://github.com/emmesgit/RSEQREP\n\nArchived source code as at time of publication: DOI is http://doi.org/10.5281/zenodo.106911445\n\nRSEQREP Amazon Virtual Machine Image available from: https://aws.amazon.com, AMI ID: RSEQREP (RNA- Seq Reports) v1.0 (ami-e1708b9b))\n\nLicense: Subject to various licenses, namely, the GNU General Public License version 3 (or later), the GNU Affero General Public License version 3 (or later), and the LaTeX Project Public License v.1.3(c).\n\nA list of the software contained in this program, including the applicable licenses, can be accessed at https://github.com/emmesgit/RSEQREP/SOFTWARE.xlsx\n\nDataset 1. RNA-Seq of PBMC and B cell gene expression profiles in healthy humans following influenza vaccination available from NCBI GEO with accession number GSE45764.\n\nDataset 2. MSigDB Version 5.2 GMT gene set files used for the influenza vaccine case study available from:\n\nhttp://software.broadinstitute.org/gsea/msigdb/download_file.jsp?filePath=/resources/msigdb/5.2/msigdb_v5.2_files_to_download_locally.zip\n\nFor MSigDB license terms, please refer to http://software.broadinstitute.org/gsea/license_terms_list.jsp. Users are requested to create a login prior to data access:\n\nhttp://software.broadinstitute.org/gsea/register.jsp?next=index.jsp\n\nDataset 3. Blood Transcription Modules GMT file used for the influenza vaccine case study available from:\n\nhttps://www.nature.com/articles/ni.2789#supplementary-information\n\n(Zip file 1).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project was funded by the Emmes Corporation and by federal funds from the National Institutes of Allergy and Infectious Disease, part of the National Institutes of Health in the Department of Health and Human Services, under Contract Nos. HHSN272200800013C and HHSN272201500002C.\n\n\nSupplementary material\n\nS1: RSEQREP Summary PDF report for influenza vaccine case study.\n\nClick here to access the data.\n\nS2: RSEQREP configuration file for influenza vaccine case study.\n\nClick here to access the data.\n\n\nReferences\n\nSboner A, Mu XJ, Greenbaum D, et al.: The real cost of sequencing: higher than you think! Genome Biol. 2011; 12(8): 125. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEmmes Git: emmesgit/RSEQREP: RSEQREP v0.9.0 (Version 0.9.0). Zenodo. 2017. Data Source" }	[ { "id": "29275", "date": "19 Jan 2018", "name": "Anup Mahurkar", "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\nRSEQREP is a comprehensive RNAseq analysis pipeline for processing bulk human RNAseq data. The pipeline bundles a number of commonly used RNAseq analysis tools to create a single pipeline for end-to-end RNAseq data analysis. The pipeline includes tools such fastqc for data QC, STAR and Hisat for alignment, RSeQC for generating alignment stats, edgeR for differential gene expression, a number of R packages for clustering analysis, and GOSeq for gene set enrichment analysis. The output from all of these tools is then used to generate a comprehensive report that summarizes the data analysis. The pipeline can be downloaded locally and run in an Ubuntu VM or can be executed on Amazon using a prebuilt Amazon Machine Image (AMI).\n\nThe article is well written with an example dataset used to illustrate the different outputs generated by the pipeline. This is a useful tool that will help small labs with limited bioinformatics expertise or computational resources run RNAseq analyses. The authors also provide documentation on the github repository. The configuration of the pipeline is made easy through an Excel template provided in the repository.\n\nWhile there are other similar tools and pipelines this is one of the few that bundles everything into a single pipeline and could be a great tool for biologist and bioinformaticians. Following are some suggestions that might improve the tool:\n\nThe authors have provided a number of useful visualizations including Heatmaps, UpSet plots, radar plots, and PCA plots, some commonly used plots in RNAseq analysis such as MA plots, or Volcano plots are not present. The authors could consider adding these plots to the package. The generated report includes summary figures for the different analyses and tables with the list of genes and pathways detected through the analyses. For a large analysis like the time-series analysis used as an example the generated report includes over 300 pages. This makes it hard to navigate through the report and find things quickly. Also, it is hard to explore the DE results by changing log-fold cutoffs, or FDR cutoffs which could easily be done in a spreadsheet but not in a PDF. The authors might want to consider splitting the report in two parts, a PDF report with images and summary data and methods, and an accompanying workbook with the tables so users can explore the results. The tool requires users to build the indexes for the database used as the reference genome. This typically requires a higher memory machine (16 GB for Hisat2, and 37 GB for STAR). The processing itself can be done with less memory. This could pose a problem for users trying to run this tool on a typical laptop or workstation. The authors could consider prebuilding some commonly used indices for human, mouse, and rat genomes so all users do not have to re-index. These references are relatively stable so the index may only need to be rebuilt once or twice a year. This will ease the burden significantly for end users. The authors have benchmarked their tool on Amazon to identify the most optimal instance type (Figure 6) so users can minimize costs. The biggest performance gains seem to be when the machine instance is changed from 4 vCPUs to 8 vCPUs. However, if a user were to want to use STAR this instance type does not have sufficient RAM. To optimize, the user will need to either use a larger instance for the entire processing, or launch two instances, a larger instance for the indexing step, and a smaller instance for processing, and copy the indices. For this reason, having pre-built indices might alleviate this issue. The documentation needs some improvement, particularly if the intended audience is users with limited computational experience. When I tried to launch the AMI on Amazon I was not sure what username to use to log into the running instance. Through trial and error, I figured out that the username was “ubuntu”. But it would be better if this were included in the documentation on the github repo. Another related issue is that because the pipeline reads Excel config files the user needs to create the config file on the local machine and upload to the AMI. Most non-tech savvy users will not necessarily know how to do it easily. The documentation could point to some utilities that could be used to upload the edited file such as sftp. It appears that the system is only setup for human genome analysis. While editing the config file it was not clear where to specify the reference genome information for other organisms. There is no reason the pipeline could not work for other model organisms which are commonly used for basic research studies. This will increase its adaption and userbase. Once I had the AMI running I had a difficult time executing the test pipeline. I was getting errors about missing directories or data files. I would recommend that the authors test the AMI and have clearer instructions on how to download datasets and run the tools in the AMI. For instance after cloning the github repo I tried running the setup script with the command “sh RSEQREP/setup.sh” while I was in “/home/ubuntu” and got the following error:\n“Fatal error: cannot open file '/home/ubuntu/source/r/parse-rnaseq-configuration.r': No such file or directory”.\n\nI then tried moving to the RSEQREP directory to run the same command and got the error:\n\n“File does not exist! /home/ubuntu/msigdb/c2.cp.kegg.v5.2.entrez.gmt.”\n\nBased on the error message it appears that the software expects the databases to be uploaded before the setup script can be run but the documentation does not specify that. As a result, I was not able to test the VM end-to-end, but with improved testing and documentation this should be easy to address.\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": "3566", "date": "13 Apr 2018", "name": "Johannes Goll", "role": "Author Response", "response": "We thank Anup Mahurkar for his insightful comments and suggestions to increase the usefulness of this software. In the following, we address each of the reviewer's comments highlighted in bold:The authors have provided a number of useful visualizations including Heatmaps, UpSet plots, radar plots, and PCA plots, some commonly used plots in RNAseq analysis such as MA plots, or Volcano plots are not present. The authors could consider adding these plots to the package.The Volcano and MA plots can be found on page Figures 20-23 in the case study report (Supplementary File S1).The generated report includes summary figures for the different analyses and tables with the list of genes and pathways detected through the analyses. For a large analysis like the time-series analysis used as an example the generated report includes over 300 pages. This makes it hard to navigate through the report and find things quickly. Also, it is hard to explore the DE results by changing log-fold cutoffs, or FDR cutoffs which could easily be done in a spreadsheet but not in a PDF. The authors might want to consider splitting the report in two parts, a PDF report with images and summary data and methods, and an accompanying workbook with the tables so users can explore the results.We recommend that users navigate to the figure and table listings at the beginning of the PDF report to find content of interest or use the PDF search option. RSEQREP provides user-friendly configuration options via a spreadsheet (see for example Supplementary File S2) including FDR and fold change cut offs that can be adjusted prior to generating reports. In addition to the PDF report, RSEQREP outputs all tables including DE gene lists in comma-separated values (CSV) format which then can be used within Excel or other spreadsheet software to dynamically filter DE gene lists.The tool requires users to build the indexes for the database used as the reference genome. This typically requires a higher memory machine (16 GB for Hisat2, and 37 GB for STAR). The processing itself can be done with less memory. This could pose a problem for users trying to run this tool on a typical laptop or workstation. The authors could consider prebuilding some commonly used indices for human, mouse, and rat genomes so all users do not have to re-index. These references are relatively stable so the index may only need to be rebuilt once or twice a year. This will ease the burden significantly for end users.We agree that a pre-built index would be more computational effective. However, we consider generating the index a part of the start-to-end analysis and, as a critical step to support reproducible research, indexing software, genome, and genome annotation versions are fully captured. This approach provides also the most flexibility to the users who can specify any Ensembl version including associated annotations and reference genome assembly during the initialization phase (see for example Supplementary File S2). A process to manually maintain genome indices would quickly become out of date.The authors have benchmarked their tool on Amazon to identify the most optimal instance type (Figure 6) so users can minimize costs. The biggest performance gains seem to be when the machine instance is changed from 4 vCPUs to 8 vCPUs. However, if a user were to want to use STAR this instance type does not have sufficient RAM. To optimize, the user will need to either use a larger instance for the entire processing, or launch two instances, a larger instance for the indexing step, and a smaller instance for processing, and copy the indices. For this reason, having pre-built indices might alleviate this issue.Since we completed our computational benchmarks, newer AWS EC2 instance types have become available. Most notably the r4.X and x1e.X instance types, which are more than capable of providing enough memory for indexing with 4 or 8 vCPUs.The documentation needs some improvement, particularly if the intended audience is users with limited computational experience. When I tried to launch the AMI on Amazon I was not sure what username to use to log into the running instance. Through trial and error, I figured out that the username was “ubuntu”. But it would be better if this were included in the documentation on the github repo.We have updated our README file on GitHub (https://github.com/emmesgit/RSEQREP). We added detailed information about the AMI, installation, execution, and troubleshooting. This includes details on which user name and password to use to login into the AMI.Another related issue is that because the pipeline reads Excel config files the user needs to create the config file on the local machine and upload to the AMI. Most non-tech savvy users will not necessarily know how to do it easily. The documentation could point to some utilities that could be used to upload the edited file such as sftp. We provide the Amazon AMI preconfigured with the X2GO remote Desktop software. This allows users to connect to the instance using a user-friendly desktop environment. We have updated our README file on GitHub (https://github.com/emmesgit/RSEQREP) to include information about this. Additionally, the instance comes pre-configured with the Libre Office software which contains an Excel editor.It appears that the system is only setup for human genome analysis. While editing the config file it was not clear where to specify the reference genome information for other organisms. There is no reason the pipeline could not work for other model organisms which are commonly used for basic research studies. This will increase its adaption and userbase. We purposefully designed the software to support human clinical research studies. While at this time, it supports only RNA-Seq analysis of human clinical samples, we may add support to expand on this in the future. We do encourage users to modify the existing source code for their own purposes including adaptations to support RNA-Seq analyses for other model organisms. Once I had the AMI running I had a difficult time executing the test pipeline. I was getting errors about missing directories or data files. I would recommend that the authors test the AMI and have clearer instructions on how to download datasets and run the tools in the AMI. For instance after cloning the github repo I tried running the setup script with the command “sh RSEQREP/setup.sh” while I was in “/home/ubuntu” and got the following error:“Fatal error: cannot open file '/home/ubuntu/source/r/parse-rnaseq-configuration.r': No such file or directory”. I then tried moving to the RSEQREP directory to run the same command and got the error: “File does not exist! /home/ubuntu/msigdb/c2.cp.kegg.v5.2.entrez.gmt.” Based on the error message it appears that the software expects the databases to be uploaded before the setup script can be run but the documentation does not specify that. As a result, I was not able to test the VM end-to-end, but with improved testing and documentation this should be easy to address.We have updated our README file on GitHub (https://github.com/emmesgit/RSEQREP). We added detailed information about using the AMI, installation, execution, and troubleshooting. We also updated the run-* scripts to be executed anywhere on the file system. Prior to running RSEQREP, most suitable gene set datasets (GTM files) for the pathway enrichment analysis (e.g. Reactome pathways, KEGG pathways, MSigDB pathways, etc.) need to be identified and downloaded by the user and added to the RSEQREP configuration file. To make it easier for users, we provide programs to download certain gene sets files (see our README file on GitHub page for further documentation)." } ] }, { "id": "29277", "date": "31 Jan 2018", "name": "Ben Busby", "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 is an important example of building and advertising easy-to-implement yet powerful bioinformatics pipelines with established, popular algorithms.\n\nThat said, in my opinion, three minor additions would make this manuscript much more robust, and therefore, acceptable for publications.\nFirst, the authors state that these pipelines should work on a variety of cloud architectures, but all they explicitly provide is an AMI for AWS use. Containerization through Docker (not an endorsement) or some other containerization protocol should simply bridge this gap.\nSecond, there are many mentions of bioconductor, but it is not clear from the manuscript whether this particular pipeline is available in bioconductor. Please clarify.\nThird, given that the components of such pipelines are continuously evolving, there should be some documentation, either in the manuscript or the github repo about switching in new modules for mapping, differential expression, or visualization.\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? 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": "3567", "date": "13 Apr 2018", "name": "Johannes Goll", "role": "Author Response", "response": "We thank Ben Busby for his insightful comments and suggestions to increase the usefulness of this software. In the following, we address each of the reviewer's comments highlighted in bold: First, the authors state that these pipelines should work on a variety of cloud architectures, but all they explicitly provide is an AMI for AWS use. Containerization through Docker (not an endorsement) or some other containerization protocol should simply bridge this gap. To increase the software's portability, we created a public RSEQREP Docker container that contains all the required 3rd party software. The container image is available at https://hub.docker.com/r/emmesdock/rseqrep and can be accessed via the Docker utility using “docker pull emmesdock/rseqrep”. Second, there are many mentions of bioconductor, but it is not clear from the manuscript whether this particular pipeline is available in bioconductor. Please clarify. RSEQREP is not available as a Bioconductor R package and includes components not written in R. The source code is available at https://github.com/emmesgit/RSEQREP Third, given that the components of such pipelines are continuously evolving, there should be some documentation, either in the manuscript or the github repo about switching in new modules for mapping, differential expression, or visualization. We agree that RNA-Seq data processing and analysis is a constantly evolving field and there is no consensus on how to best analyze the data. RSEQREP includes a collection of best practice analytical tools that we identified through extensive review of the peer-reviewed literature. Users have complete access to the RSEQREP source code to make custom updates to all workflow, analysis, and reporting components. In combination with scalable cloud resources this allows for rapid prototyping of analysis reports. Below are key RSEQREP programs to alter mapping, differential expression, visualizations, or tables: 1) Read mapping is executed via “RSEQREP\\source\\perl\\preprocess-rnaseq.pl” 2) DE gene analysis is executed via “RSEQREP/source/r/02-sdeg-identification/init-edgeR-glm-model.r” 3) Integration of visualizations (R graphics) as part of the report is handled via “/RSEQREP/source/knitr/figures.Rnw” files. 4) Integration of tables (R xtable) as part of the report is handled via “/RSEQREP/source/knitr/tables.Rnw” files." } ] } ]	1	https://f1000research.com/articles/6-2162
https://f1000research.com/articles/7-449/v1	11 Apr 18	{ "type": "Research Article", "title": "Educational sessions may not be enough to improve knowledge about hand hygiene: Assessing the knowledge about hand hygiene of health workers before and after an educational workshop in Sudan", "authors": [ "Ihab B. Abdalrahman", "Sara Shamat", "Sara Mamoun", "Reem Abdelraheem", "Esraa Salah", "Mohammed Elkhalifa", "Abduraheem Farah", "Duaa Ali", "Abdelmohaymin A. Abdalla", "Eman Saeed", "Mohamed Dafaalla", "Ihab B. Abdalrahman", "Sara Shamat", "Sara Mamoun", "Reem Abdelraheem", "Esraa Salah", "Mohammed Elkhalifa", "Abduraheem Farah", "Duaa Ali", "Abdelmohaymin A. Abdalla", "Eman Saeed" ], "abstract": "Background: In an attempt to defeat the high prevalence of health care associated infections, by raising the awareness about hand hygiene, we implemented a quasi-experimental study as part of a quality improvement project to evaluate the efficacy of focused educational workshop on knowledge about hand hygiene among health care workers in Sudan. Methods: Seventy-three participants were recruited from two public hospitals and one private hospital in Khartoum, Sudan in September 2017. The knowledge before and after the educational workshop was assessed for each participant using the World Health Organization hygiene associated infection questionnaire. We analyzed data using SPSS v22 and used McNemar’s test to compare knowledge before and after the workshop. Results: Around 40% of participants worked in general ward and 85% of them were women. Almost 70% were midwives or nurses. The mean age of participants was 28.4 years. We compared the knowledge of hand hygiene between nurses and doctors before the workshop, and the results showed that nurses had better knowledge in almost all aspects of hand hygiene. When we compared the knowledge before and immediately after the workshop for all participants, we found that there was no significant improvement in almost all aspects of knowledge about hand hygiene (P>0.05). Conclusion: Comparing the knowledge before and after the workshop, we found no significant improvement in almost all aspects of hand hygiene. Of note, nurses’ knowledge about hand hygiene was consistently higher than doctors. Additional studies are needed to identify the optimal design of educational sessions.", "keywords": [ "Educational sessions", "hand hygiene", "infection control", "nurses and doctors", "workshop", "health-care associated infections" ], "content": "Introduction\n\nHealth-care associated infections (HAIs) are regarded as a major health problem endangering hospital-admitted patients in particular1. In developed countries, 5–15% of hospitalized patients were at risk to acquire infections, especially those admitted to intensive care units; HAIs in Europe and USA were 4.6–9.3% and 4.5%, respectively2. In contrast, the prevalence in some developing countries is as high as (19.1–14.8%)3. Health care associated infections (HAIs) results in prolonged hospital stay as it can add 18–24 days to the length of hospital stay and may result in more deaths. For instance, in Europe HAIs are responsible for 50,000–135,000 deaths out of 5,000,000 cases of HAIs, annually4. Moreover, it contributes to €13–24 billion of added healthcare costs5. All these facts highlight the morbidity and mortality from HAI.\n\nHand hygiene is a general term, referring to any action of hand cleansing using water and detergent and/or the use of alcohol-based hand sanitizers for the removal of transient microorganisms from hands6. It is widely accepted that improved hand hygiene compliance contributes to the prevention of HAIs7. This consensus has been supported by several studies. For example, a hand hygiene culture changing program was conducted by Grayson et al. (2008) over two years in six Australian hospitals. This study demonstrated that the incidence of Methicillin-resistant-Staphylococcus aureus (MRSA) bacteraemia and the number of MRSA-positive clinical isolates were significantly reduced at the end of the study period5. Hand hygiene is found to be the single most effective measure to guard against healthcare-associated pathogens8.\n\nThere are various educational methods adopted to improve knowledge about hand hygiene as a key element to control HAI. For instance knowledge about hand hygiene could be included in the curriculum for medical and nursing students9. Another way is by taking advantage and to introduce the appropriate knowledge about hand hygiene to health care professionals gathered at conferences and professional meetings8. In addition educational workshops and sessions about hand hygiene among hospital staff should be arranged from time to time8. Lastly, educational hand-outs and posters about hand hygiene should be available in the working environments of health workers1.\n\nImplementation of different approaches regarding boosting hand hygiene knowledge shows a wide range of variability in the efficacy between educational and interventional approaches. Hand hygiene compliance improvement has been shown to be greater using educational approaches than practical interventions10. For instance, after delivery of an educational hand-out and poster campaign the rate of study participant complying with hand-washing guidelines was 83%1. In addition, after two years of commencement of The Australian National Hand Hygiene Initiative in 2009, hand hygiene compliance increased from 43.6% to 67.8%7. Thus, giving educational lectures and workshops about hand hygiene practice appeared to have a noticeable impact on the knowledge and practice of hand hygiene among health care workers in these settings.\n\nThis study aimed to evaluate the knowledge about hand hygiene among heath care workers in Sudan. In addition, we assessed the role of an educational session as a recommended tool to improve the knowledge about hand hygiene among these same health care workers.\n\n\nMethods\n\nEthical approval was obtained from Soba University Teaching Hospital and Soba Centre for Audit and Research (approval no S248). All participants were fully informed about the workshop and the study prior to participation. Written consent was obtained from all participants for participation.\n\nWe implemented a quasi-experimental study as part of a quality improvement project designed and implemented by Soba University Hospital, University of Khartoum, to evaluate the efficacy of focused educational workshop on knowledge about hand hygiene among health care workers.\n\nAll health care providers in departments of emergency and internal medicine (73 participants) were recruited from two public hospitals (Soba Teaching Hospital and Saad Abuelela Hospital) and one private hospital (Fedail Hospital) in Khartoum, Sudan, in September 2017. Participants were recruited by the departments of infection control in their hospitals to enrol in this quality improvement project. Their knowledge before and after the educational workshop (see below) was assessed using the World Health Organization (WHO) HAI questionnaire.\n\nThe questionnaire is composed of 20 single best answer questions. The first seven questions assess demographic variables; hospital name, ward, age, gender, profession, and previous hand hygiene training. The remaining questions assess different aspects of knowledge about hand hygiene11.\n\nThe educational workshop was composed of lectures and practical sessions delivered over eight working hours (see Supplementary File 1). The workshop was delivered by a qualified instructor who has completed a PhD on infection control. The knowledge about hand hygiene measures was delivered mainly through lectures, while the practical sessions focused on training about proper hand washing technique according to the WHO11.\n\nWe analyzed data using SPSS v22. We used McNemar’s test to compare knowledge of participants before and after the educational workshop\n\n\nResults\n\nAround 40% of participants worked in general ward and 85.9% of them were women. Almost 70% were midwives or nurses. The mean age of participants was 28.4 years. Table 1 shows the demographic characteristics of the participants.\n\nWe compared the knowledge of hand hygiene between nurses and doctors, and the results showed that nurses had a better knowledge in almost all aspects of hand hygiene. When we compared the knowledge immediately after the workshop we found out that there was no significant improvement in almost all aspects of knowledge about hand hygiene (P>0.05). Table 2 illustrates these findings in more details. The text in bolds are the correct answers. P values in bold are the significant probabilities.\n\n(A) Hand disinfection topics; (B) hand rubbing, hand washing, and accessories topics. We used McNemar’s test to compare knowledge of participants before and after the educational workshop. P values <0.05 indicates significant difference.\n\n\nDiscussion\n\nOur study revealed that knowledge about hand hygiene and HAI is better among nurses and midwifes in comparison to doctors. Some studies concluded the same results. In a study conducted by Ameer et al. nurses were found to have a better hand hygiene compliance rate (43.08%) compared to doctors (31.25%). Another study by Han et al. revealed that nurses’ knowledge score was significantly higher than doctors12. On the other hand, other studies showed opposite results. For example, Ekwere et al. conducted a study in a tertiary hospital in Southwest Nigeria and concluded that doctors had no significant better knowledge of hand washing than nurses13.\n\nOverall there was no significant improvement in knowledge about hand hygiene and HAI after the educational workshop. Similarly, in a study done by Lee et al. there was no significant improvement in hand hygiene compliance or alcohol-based hand rub consumption following education10. In contrast, a case–control study that evaluated the effect of using educational activities and posters on hand hygiene compliance revealed a significant improvement in hand hygiene compliance compared to control hospitals8. Similarly, Abdraboh et al. (2012) concluded that performing educational sessions was among the most important activities to attain better health care worker hand hygiene compliance3.\n\nBut why didn’t we improve hand washing knowledge after administration of the workshop? This could be attributed to deficiency in contents, environment, and teaching methods. Regarding teaching methods; the lecture duration was 3 hours which may not only make some of the candidates fail to follow the instructor but may also make it difficult for the instructor to stay focused. Moreover, the large number of participants (n=73) might make it difficult for instructors to deliver effective education. The present result might be of help to alert instructors to review their teaching methods and these results emphasize the importance of feedback in improving learning methods. Cook in the article ‘Twelve tips for evaluating educational programs’ strongly advises that instructors should seek evaluation from stakeholders such as students and administrators. In addition, designing and validating an evaluation tool to evaluate the quality and effectiveness of educational programs would be of great help because it enables reliable evaluation and monitoring of the progress of the program14.\n\n\nConclusions\n\nIn conclusion, to meet the objectives of educational workshops we recommend that contents should be revised and cover all the hand hygiene guidelines that are stated by WHO. In addition, taking into consideration the language factor in delivering the information in understandable language to all participants. Lastly, the number of participants as well as the duration of the workshop should be reduced to a level that facilitates proper information delivery.\n\n\nData availability\n\nDataset 1: Participant responses to the WHO handwashing questionnaire data before and after the educational session. DOI 10.5256/f1000research.13029.d19968715", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that there were no grants involved in supporting this study.\n\n\nSupplementary material\n\nSupplementary File 1: Educational workshop content.\n\nClick here to access the data.\n\n\nReferences\n\nCreedon SA: Healthcare workers' hand decontamination practices: compliance with recommended guidelines. J Adv Nurs. 2005; 51(3): 208–216. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Improved hand hygiene to prevent healthcare-associated infections. Patient Safety Solutions, [online] 2007; 1(Solution 9). Reference Source\n\nAbdraboh SN, Milaat W, Ramadan IK, et al.: Hand hygiene and health care associated infection: an intervention study. American Journal of Medicine and Medical Sciences. 2016; 6(1): 7–15. Reference Source\n\nWHO Guidelines for Hand Hygiene in Health Care: First Global Patient Safety Challenge: Clean Care is Safer Care. Reference Source\n\nvan De Mortel TF, Kermode S, Progano T, et al.: A comparison of the hand hygiene knowledge, beliefs and practices of Italian nursing and medical students. J Adv Nurs. 2012; 68(3): 569–579. PubMed Abstract \| Publisher Full Text\n\nNabavi M, Alavi-Moghaddam M, Gachkar L, et al.: Knowledge, Attitudes, and Practices Study on Hand Hygiene Among Imam Hossein Hospital's Residents in 2013. Iran Red Crescent Med J. 2015; 17(10): e19606. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcLaws ML: The relationship between hand hygiene and health care-associated infection: it's complicated. Infect Drug Resist. 2015; 8: 7–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMathai E, Allegranzi B, Seto WH, et al.: Educating healthcare workers to optimal hand hygiene practices: addressing the need. Infection. 2010; 38(5): 349–356. PubMed Abstract \| Publisher Full Text\n\nOjulong J, Mitonga KH, Iipinge SN: Knowledge and attitudes of infection prevention and control among health sciences students at University of Namibia. Afr Health Sci. 2013; 13(4): 1071–1078. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Y, Wang Y, Yan D, et al.: Self-reported hand hygiene practices, and feasibility and acceptability of alcohol-based hand rubs among village healthcare workers in Inner Mongolia, China. J Hosp Infect. 2015; 90(4): 338–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Clean Care is Safer Care, tools for evaluation of hand hygiene practices and related perception and knowledge at a health-care facility is one vital element of the strategy to improve hand hygiene. Reference Source\n\nHan K, Dou FM, Zhang LJ, et al.: [Compliance on hand-hygiene among healthcare providers working at secondary and tertiary general hospitals in Chengdu]. Zhonghua Liu Xing Bing Xue Za Zhi. 2011; 32(11): 1139–1142. PubMed Abstract \| Publisher Full Text\n\nEkwere TA, Okafor IP: Hand hygiene knowledge and practices among healthcare providers in a tertiary hospital, south west, Nigeria. Int J Infect Control. 2013; 9(4). Publisher Full Text\n\nCook DA: Twelve tips for evaluating educational programs. Med Teach. 2010; 32(4): 296–301. PubMed Abstract \| Publisher Full Text\n\nAbdalrahman IB, Shamat S, Mamoun S, et al.: Dataset 1 in: Educational sessions may not be enough to improve knowledge about hand hygiene: Assessing the knowledge about hand hygiene of health workers before and after an educational workshop in Sudan. F1000Research. 2018. Data Source" }	[ { "id": "34546", "date": "25 Jun 2018", "name": "Sile A. Creedon", "expertise": [ "Reviewer Expertise My area of research is in infection prevention in acute care settings" ], "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 paper. I hope my comments are useful to you.\nIntroduction: Can you check the accuracy of the figures related to HCAI in developing countries please.\nIntervention: The design of the educational intervention was face to face workshop. Given that this was the intervention, it needs to be described in much more detail. What other types of educational interventions were considered and why was this type chosen? Can you please add a referenced section to the introduction.\n\nDesign: The study design was quasi experimental. The authors need to explain more fully how the study met the criteria for quasi experimentation. Could it be more accurately described as an interventional study?\n\nSample: The participants were chosen by the infection prevention staff in each hospital. How did this occur? it needs a better explanation and the threat of bias also needs to be discussed. It seems to me that this may lead to targeting only staff who were 'known to' or perhaps were friendly with the infection prevention team.\n\nDiscussion: The discussion section needs to be developed more fully. In the opening paragraph, the authors state that there was no improvement in either knowledge or prevalence of HCAI's after the workshop. Did the study measure prevalence of HCAI's before and after the work shop? If not, then you might review making this statement.\n\nThere is an attempt made to elucidate why the intervention did not work but it needs more than this. It would benefit from discussion of the design. Is there value in assessing knowledge immediately post intervention? Did you consider a longitudinal design?\n\nOverall, there is some merit in this paper but it needs further development.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "135018", "date": "04 May 2022", "name": "Caitlin Liddelow", "expertise": [ "Reviewer Expertise Behaviour change", "health psychology", "health interventions" ], "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 would like to thank the editor and the authors for allowing me the opportunity to review this paper. It addresses an important topic, hand hygiene in healthcare, which has been of particular interest the last few years. The paper is mostly well-written (some minor grammatical and language expressions that could be improved), however I am concerned by the lack of critical thinking and discussion in the introduction and discussion. There is a lot of behaviour change literature out there, lots with a focus on hand hygiene behaviours, which do not appear in this paper. There are also some details in the methods and results that should be included. I believe once my below comments have been addressed, the paper will be much improved.\nAbstract:\nThe abstract is clear and presents all of the necessary information required for an abstract.\n\nIntroduction:\n\nPage 3: Please provide some examples of health-care associated infections, for readers that may not be in health-care specifically.\n\nPage 3: For consistency, please remove the second (HAIs) from the first paragraph, as you have already done this in the first sentence.\n\nPage 3: Given the experimental study is based in Sudan, are there any statistics or figures regarding the prevalence of HAIs or HAI associated deaths in Sudan? This would be good to include in the introduction.\n\nPage 3: Your reference/citation for this sentence/study is incorrect. You talk about an Australian study by Grayson et al. but there is not Grayson et al. in your reference list. Please fix this: “For example, a hand hygiene culture changing program was conducted by Grayson et al. (2008) over two years in six Australian hospitals. This study demonstrated that the incidence of Methicillinresistant-Staphylococcus aureus (MRSA) bacteraemia and the number of MRSA-positive clinical isolates were significantly reduced at the end of the study period”.\n\nPage 3: Whilst you provide a good overview of the current literature in the hand hygiene area, there have been numerous studies that have come out since the beginning of the Covid-19 pandemic that look at hand hygiene interventions. It would be good to see some of these studies cited in your introduction.\n\nPage 3: Your introduction lacks some critical thinking and review. I am curious as to why you have decided to do an experimental study on the impact of an education session on hand hygiene when the previous literature already shows this is effective? What is the reason for doing this study given these past studies? Is it because there are differences in the Sudanese culture that may not yield the same results as other studies? More of a rationale for why you have decided to do this specific study is needed.\n\nMethods:\nPage 3: Please provide an example question from the WHO hygiene questionnaire.\n\nPage 3: How was this questionnaire scored? Was it a score out of 20? Did participants get separated into groups (e.g., low knowledge / high knowledge)?\n\nPage 3: Some more information on the workshops would be useful. Was it all delivered in one day? Was it in person or online? Was it conducted in one large group at each hospital, or multiple smaller groups? Where was the content in the workshops/lectures derived from? Did the practical sessions involve watching videos, role play, etc.? If possible, matching some of the components of the workshop to Behaviour Change Techniques (see Michie et al., 20131) would be useful.\n\nPage 3: You say you “used McNemar’s test to compare knowledge of participants before and after the educational workshop” but on page 4 under “multivariate analysis” you say that you compared between groups (doctors vs. nurses). This needs to be clearer on page 3.\n\nPage 3: Why was McNemar’s test conducted rather than a paired samples t-test (for before vs. after) or independent samples t-test (for doctors vs. nurses)? It is unclear why you would use a McNemars test (which is used with nominal data) when it appears, or is assumed, the score from the WHO questionnaire is scale data. More information and a rationale is required.\n\nResults:\nPage 3/4: Did you collect information on how long participants had been in the profession? I would expect this would be important to know as it likely influences the results (i.e., if they have worked longer, they are more likely to have higher knowledge).\n\nPage 4/5: Please be consistent with your reporting of percentages and p-values. Some have 1 decimal place, some have 2 decimal places and some have 3.\n\nPage 4: It would be nice to see a combined hand hygiene knowledge score/percentage correct of participants from before the workshop to after the workshop.\n\nDiscussion:\nPage 6: Please include a correct citation and reference for Ameer et al. and Han et al. in the first paragraph of your discussion. You should not be discussing studies that you have not properly referenced.\n\nPage 6: It would be good if you included further discussion on why you/the authors think you received the results you did. In the first paragraph you provide evidence that is inline with your finding and then evidence that is the opposite. Why do you think nurses and midwives had greater knowledge compared to doctors?\n\nPage 6: There is an abundance of literature and research that says that providing education alone is not enough to change behaviour. This may be why you did not see any significant improvements after the workshop, as it was only education based. Some discussion of this is warranted, particularly in paragraph 2. See: Arlinghaus K. R., & Johnston, C. A. (2017). Advocating for Behavior Change With Education2. Page 6: Please fix this in-text citation “Cook in the article ‘Twelve tips for evaluating educational programs’.\n\nPage 6: More discussion of the theoretical and/or practical implications of this study is needed. For example, what do the findings tell us about hand hygiene knowledge amongst this sample, specifically in Sudan? What does it tell us about the difference between nurses and doctors?\n\nPage 6: There is no mention of strengths or limitations, or any suggestions for future research in this area. Please consider including some.\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? 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-449
https://f1000research.com/articles/7-287/v1	07 Mar 18	{ "type": "Research Article", "title": "The acid tolerance response and pH adaptation of Enterococcus faecalis in extract of lime Citrus aurantiifolia from Aceh Indonesia", "authors": [ "Zaki Mubarak", "Cut Soraya", "Cut Soraya" ], "abstract": "Background: The objective of the present study was to evaluate the acid tolerance response and pH adaptation when Enterococcus faecalis interacted with extract of lime (Citrus aurantiifolia). Methods: We used E. faecalis ATCC 29212 and lime extract from Aceh, Indonesia. Both materials were analyzed for their pH adaptation, acid tolerance response, adhesion assay, and mass profiles using a light microscope with a magnification of x1000. Further, statistical tests were performed to analyze both correlation and significance of the acid tolerance and pH adaptation also the interaction activity. Results: E. faecalis was able to adapt to a very acidic environment (pH 2.9), which was characterized by an increase in its pH (reaching 4.2) at all concentrations of the lime extract (p < 0.05). E. faecalis was also able to provide acid tolerance response to lime extract based on spectrophotometric data (595 nm) (p < 0.05). Also, the interaction activity of E. faecalis and the lime extract was relatively stable within 6 up to 12 hours (p < 0.05), but it became unstable within 24–72 hours (p > 0.05) based on the mass profiles of its interaction activity. Conclusions: E. faecalis can adapt to acidic environments (pH 2.9–4.2); it is also able to tolerate acid generated by Citrus aurantiifolia extract, revealing a stable interaction in the first 6–12 hours.", "keywords": [ "Enterococcus faecalis", "Lime (Citrus aurantiifolia) extract", "acid tolerance", "pH adaptation" ], "content": "Introduction\n\nEnterococcus faecalis is a significant agent in the pathogenesis of root canal infections, especially in post-endodontic treatment, with a prevalence of 24–77% in these infections1. E. faecalis is very difficult to eliminate because the pathogen can survive in poor nutrient conditions. It can adapt to acidic conditions, including living in the dentin tubule of a closed root canal with a smear layer. It can also express the dominant biofilm protein to maintain its attachment to host cells2.\n\nE. faecalis has been shown tolerate to acidic environments as well as to adapt to pH changes, which are the essential virulence factors in maintaining antibacterial balance3. Fisher reported that E. faecalis could survive in environments with high NaCl concentrations at extreme temperatures of 5–65°C with a pH of 4.5–10.04. Stuart et al.1 reported that E. faecalis are less sensitive, with a pH of 5.0 at 25°C after it has been incubated for 10 h. The author also found that it has an excellent growth capability at pH 8.5 and low adhesion at pH 7.1 in a medium coated with bovine serum albumin (BSA).\n\nE. faecalis is resistant to medication materials such as calcium hydroxide5 and chlorhexidine (CHX)6. The long-term use of both medication materials can lead to parachloroaniline (PAC), causing blockage of the dentinal tubules and eventually becoming toxic7. Fosfomycin may also interfere with acid tolerance systems and pH changes of E. faecalis in tooth root canals by inhibiting phosphoenolpyruvate synthetase8.\n\nIndonesia, especially in Aceh has a tropical climate with a variety of plants that can be utilized in medical treatment, including lime extract (Citrus aurantiifolia). It contains phenols, flavonoids, hydrogen peroxide, tannins, alkaloids, and saponins that have antibacterial, antioxidant, antifungal, analgesic, and anti-inflammatory properties9. Nwankwo10 reported that lime extract helped to prevent Klebsiella pneumonia, Salmonella, and Escherichia coli. Here, the acid in lime extract influenced the bacterial development and cell metabolism. The present study evaluates the acid tolerance response and pH adaptation of E. faecalis when it interacts with lime extract.\n\n\nMethods\n\nThe lime extract and E. faecalis (ATCC-29212) were used in this study. The extractions were prepared at the Laboratory of Microbiology at the Faculty of Veterinary, University of Syiah Kuala, Darussalam, Banda Aceh, Indonesia. Both materials were made in vitro to analyze the pH adaptation, acid tolerance response, and interaction activity between E. faecalis and lime extracts.\n\nLime peel was separated from the flesh then dried using dehydrator until the water content reduced to 10%. Dried lime peel was grinded into powders. The powder was put into glass container and masserated with ethanol 70% for two days and then strained using a gauze. Filtrate was evaporated using a rotary evaporator at 80°C to obtain the pure lime extracts.\n\nE. faecalis ATCC 29212 taken from glycerol stock was cultured on a Mueller-Hinton Agar (MHA) medium at a temperature of 80°C (Thermo Fisher Scientific Inc., Paisley, UK). The culture was incubated in anaerobic conditions at 37°C for 48 hours using Anaerogen TM GasPack (Oxoid, Basingstoke UK), and incubator (Memmert, Germany). A colony of E. faecalis bacteria was subsequently re-cultured in 5 ml of Mueller-Hinton Broth (MHB) medium (Thermo Fisher Scientific Inc, Paisley, UK) in anaerobic conditions at a temperature of 37°C for 48 hours. Afterward, the E. faecalis grown on the liquid medium was synchronized further with McFarland 0.5 (1 x 108 CFU/ml) (TM50, Dalynn Biological Inc., Calgary, Canada). The Accurate of the density of McFarland standart can be checked using a spectrophotumeter with an absorbance reading of 0.08 to 0.1 at 625 nm11.\n\nA total of 50 ml of lime extracts in several different concentrations (100%, 75%, 50%, 25%, 12.5%, and 6.25%) was placed into different beaker glasses. Then, 5 ml of E. faecalis in MHB (1:10) were added to each of the beakers. The initial pH of the mixture was measured (0 hours) before incubation. Next, each beaker was incubated at 37°C for 6 hours, 12, hours, 24 hours, 48 hours, and 72 hours in an anaerobic atmosphere using Anaerogen TM GasPack (Oxoid, Basing stoke UK), at each of these times, the beakers' pH was measured using a pH meter (Thermo Fisher Scientific Inc, Paisley, UK). Various changes in pH from 0 hours to the specified time can be used as an indicator of whether E. faecalis has a tolerance response to the acidic environment and can adapt to changing pH12.\n\nThe cultures of the pH measurements were used to measure the acid tolerance response of E. faecalis to lime extract utilizing the principle of spectrophotometry13. The analysis was performed based on the incubation time that had been determined following the measurement of pH shaken at 500 rpm. Here, 96 wells of the triple microplate series were coated with 50 μl of MHB (Thermo Fisher Scientific Inc., Paisley, UK) for 15 minutes and then were vacuumed. After that, 100 μl of the test materials (E. faecalis + lime extract) derived from the incubation processes at 6 h, 12 h, 24 h, 48 h, and 72 hours were added to each well. Each well was incubated for 15 minutes to analyze the adaptation of the test materials. Then bio-tolerant activity was measured using the Elisa Reader (Bio-Rad Laboratories, Hercules, CA) at a wavelength of 595 nm.\n\nAdhesion assay was analyzed based on the principles of Gram-staining14. The standard protein concentration of the E. faecalis and the active component concentration of the lime extract were measured via the Bradford method (Bio-Rad, Hercules, California, U.S.A.) using Bovine serum albumin (Merck, Darmstadt, Germany) as the standard protein. Spectrophotometry detected the interaction of E. faecalis with lime extract at a wavelength of 595 nm13,15.\n\nThe principle of incubation time-based interaction activity on the microplate 96 wells series used in this research based on Gamble's working principle16; it was modified using violet crystalline and safranin staining. First, 96 wells of the triple microplate series were coated with 50 μl of MHB (Thermo Fisher Scientific Inc., Paisley, UK), settled for 15 minutes, and then aspirated. Second, 50 μL of E. faecalis was added and then incubated for 15 minutes at room temperature. Third, 100 mL of the lime extract was added and incubated for 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours (as adapted from research conducted by Bachtiar)17. All of the residues of the test materials (E. faecalis + lime extract) in the microplate wells were aspirated and then settled for 10 minutes at room temperature. Then, 50 μL of 2% violet crystalline were added to each well for 5 minutes; the wells were washed with phosphate buffer saline (PBS) two times (Merck, Darmstadt, Germany).\n\nA total of 100 μL of Lugol solution was added for 1 minute and then washed with PBS. The rest of the cell metabolism that was not bacterial cells was dissolved in 96% alcohol for 20 seconds until the dye completely removed. 50 μL of safranin solution was added for 2 minutes and then washed again with PBS18. The interaction activity between the lime extract and the E. faecalis bacteria in the microplate wells was assessed via an Elisa reader using a spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 595 nm13.\n\nThe anti-adhesion mass of lime extract against E. faecalis that formed on each base of the microplate wells was prepared by adding 100 μl of glycerol for 24 hours to maintain moisture. Visualization was produced by adding 10 μl of immersive oil to each microplate well to be observed under a light microscope at x1000 magnification (Olympus CX21 FS 1, Japan) supported by Optilab Viewer software V 2.2 (Miconus Transdata Nusantara, Jakarta, Indonesia) adapted from a study conducted by Gani19.\n\nE. faecalis acid tolerance and adhesion to lime extract were calculated to determine average values and standard deviations for each concentration. Two-ways analysis of variance (ANOVA) was performed with significance set at p < 0.05. The analysis was performed using SPSS ver. 20.0 software.\n\n\nResults\n\nThe experiment was conducted in three replicates. The ANOVA test showed that lime extracts and exposed time gave the significant effect on the pH, optical density of acid tolerance respond of E. faecalis in lime extract and optical density of adhesion of interaction activity between E. faecalis and lime extract on biofilm (p<0.05). In addition, the interaction between lime extract and exposed time also gave the significant effect on the pH, optical density of acid tolerance respond of E. faecalis in lime extract and optical density of adhesion of interaction activity between E. faecalis and lime extract on biofilm (p<0.05). The results showed that E. faecalis possessed the ability to adapt to acidic (pH <7) and alkaline circumstances (pH 7>) (Figure 1). Also, E. faecalis is tolerant in the acid of lime extract with different intensities at each concentration (Figure 2). Figure 3 shows that the interaction activity of E. faecalis in lime extract influenced by time and concentration were significant (p<0.01) with strong correlation (0.98). The results showed that the interaction activities of E. faecalis in Lime extract will decrease from 6, 12, 24, and 72 hours.\n\nE. faecalis did not express an ability to adapt to the acidic pH of lime extract after interacting with fosfomycin (as a positive control) (Figure 1), although it still expressed acid tolerance response (Figure 2). Its interaction activity was robust (Figure 3). The mass profile also indicated interaction activity between lime extract and E. faecalis. Antibiotics are capable of forming a covalent bond to activate the cysteine residue of a bacterial cell, triggering UDP-N-acetylglucosamine to form hydrogen bonds. It inhibits the synthesis of peptidoglycan as an antibacterial defense.\n\n\nDiscussion\n\nAccording to Sitanggang et al.20, the lime extract has a highly acidic pH ranges (1.7–3.1). The acidity is generated by citric acid and amino acids, while the essential oils contribute to maintaining its acidic pH21. Citric acid is reported to play a crucial role as a natural material to maintain pH balance and possesses antibacterial activity22. Figure 1 shows an acidic pH of 2.9 on the extracted bar (without E. faecalis). After E. faecalis was added at various concentrations, there was a significant change (increase) in the acidic pH of lime extract (as a negative control) (p < 0.05). The increased acidic pH of lime extract from 6.25% (µg/ml) to 100% (µg/ml) (see Figure 1) indicates that E. faecalis can adapt to environments with an acidic pH (2.9–4.2) at a temperature of 37°C. Morandi reported that E. faecalis could adapt to situations with a low pH and temperature, although they become less sensitive; however, when adjusted to a pH of 5.0 at 25°C, they display increased sensitivity within 10 hours23,24.\n\nE. faecalis had an acid tolerance response to lime extract that significantly increased as the concentration of the lime extract increased (Figure 2) (p < 0.05). The increased acid tolerance response correlates with the characteristics of Enterococcal strains producing lipoteichoic acids that contribute to biofilm formation and have resistance to antibacterial agents, including tolerance to acidic environmental change25. Molecularly, the acid tolerance response of E. faecalis is influenced by the EfCitH gene, which encodes the citrate transporter protein on the surface of the cell membrane that acts to maintain the balance of the effects of citric acid generated from the environment26. Sarantinopoulos found that enterococcal strains have metabolic potential against the citrate metabolism; this supports their acid tolerance response to environmental influences such as aroma and fermentation products27. In this research, Fosfomycin with a pH of 7.2 (Figure 1) could still slightly tolerate the acidic effects. The acid tolerance response is related to the ability of E. faecalis to grow in environments with an alkaline pH (9.5–12) within 48–72 hours12.\n\nIn general, the interaction activity between E. faecalis and lime extract at all concentrations was lower than the interaction activity between E. faecalis and fosfomycin. Due to its size, the lime extract was considered to be stable based on its treatment concentration (Figure 3). Based on the incubation time, the interaction activity between E. faecalis and lime extract at all concentrations within 6–12 hours at the temperature of 37°C was also relatively stable (Figure 3). Varoni et al.28 reported that anti-adhesion activity between plant polyphenol-rich extract and Streptococcus mutans bacteria was at its maximum within 24 hours, while within 6, 7, and 8 hours, the activity was stable but not yet maximal.\n\nFigure 3 shows there was a significant increase in the average concentration bar (p > 0.05), with a relatively high error bar (standard deviation value) within 24–72 hours. It indicates that from 24–72 hours, E. faecalis begins to adapt and tolerate the temperature and pH of the environment. The mechanism utilized by bacteria to survive heat and low-pH of the environment operate in many different ways. The most successful means of surviving low-pH stress is the complete avoidance of extremely acidic environments. However, none more critical than the sensing of mild acidification to prevent the potentially lethal consequences of the inappropriate production of potentially antigenic proteins. Bacteria that are forewarned by mild acidification can prepare through the induction of a wide range of protective measures. It can alter the composition of the cell membrane, extrude protons, protect macromolecules, alter metabolic pathways, and generate alkaline29.\n\nThe interaction activity between lime extract and E. faecalis can be assumed to be the antibacterial activity, because there was a decrease in the interaction activity between E. faecalis and the biological components of lime extract within 6–24 hours and again within 48–72 hours. The interaction activity is related to the activity of the active ingredients contained in the lime extract, such as flavonoids (polyethoxylated flavones and flavanones), coumarin, and terpenoids, all of which act as antibacterials30,31.\n\n\nConclusion\n\nE. faecalis can adapt to environments with a pH of 2.9–4.3 generated by lime extracts. In addition E. faecalis also expressed a tolerance response to the acidic environment. The interaction activity between E. faecalis and lime extract become stable within 6–12 hours at a temperature of 37°C. Therefore, the lime extract can be used to inhibit the E. faecalis growth.\n\n\nData availability\n\nDataset 1: pH adaptation of E. Faecalis in lime extract based on replications 10.5256/f1000research.13990.d19664332.\n\nDataset 2: Optical density (OD) of acid tolerance respond of E. Faecalis in lime extract based on replications 10.5256/f1000research.13990.d19664433.\n\nDataset 3: The OD value of the interaction activity of E. Faecalis in lime extract based on replications 10.5256/f1000research.13990.d19664634.", "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\nWe would like to thank the Laboratory of Microbiology at the Faculty of Veterinary, Syiah Kuala University, Darussalam, Banda Aceh, Indonesia for preparing the E. faecalis ATCC 29212 and lime extract as the test materials used in this study.\n\n\nReferences\n\nStuart CH, Schwartz SA, Beeson TJ, et al.: Enterococcus faecalis: Its role in root canal treatment failure and current concepts in retreatment. J Endod. 2006; 32(2): 93–8. PubMed Abstract \| Publisher Full Text\n\nSaber S, El-Hady SA: Development of an intracanal mature Enterococcus faecalis biofilm and its susceptibility to some antimicrobial intracanal medications; An in vitro study. Eur J Dent. 2012; 6(1): 43–50. PubMed Abstract \| Free Full Text\n\nMorente EO, Fernández-Fuentes MA, Burgos MJ, et al.: Biocide tolerance in bacteria. Int J Food Microbiol. 2013; 162(1): 13–25. PubMed Abstract \| Publisher Full Text\n\nFisher K, Phillips C: The ecology, epidemiology and virulence of Enterococcus. Microbiology. 2009; 155(6): 1749–57. PubMed Abstract \| Publisher Full Text\n\nDistel JW, Hatton JF, Gillespie MJ: Biofilm formation in medicated root canals. J Endod. 2002; 28(10): 689–93. PubMed Abstract \| Publisher Full Text\n\nArias-Moliz MT, Ferrer-Luque CM, Espigares-García M, et al.: Enterococcus faecalis biofilms eradication by root canal irrigants. J Endod. 2009; 35(5): 711–14. PubMed Abstract \| Publisher Full Text\n\nKandaswamy D, Venkateshbabu N, Gogulnath D, et al.: Dentinal tubule disinfection with 2% chlorhexidine gel, propolis, morinda citrifolia juice, 2% povidone iodine, and calcium hydroxide. Int Endod J. 2010; 43(5): 419–23. PubMed Abstract \| Publisher Full Text\n\nMichalopoulos AS, Livaditis IG, Gougoutas V: The revival of fosfomycin. Int J Infect Dis. 2011; 15(11): e732–e39. PubMed Abstract \| Publisher Full Text\n\nLoizzo MR, Tundis R, Bonesi M, et al.: Evaluation of Citrus aurantifolia peel and leaves extracts for their chemical composition, antioxidant and anti-cholinesterase activities. J Sci Food Agric. 2012; 92(15): 2960–67. PubMed Abstract \| Publisher Full Text\n\nNwankwo IU, Ekpe IN, Osaro-Matthew RC: Synergistic antibacterial potentials of Citrus aurantifolia (lime) and honey against some bacteria isolated from sputum of patients attending Federal Medical Center Umuahia. Int J Curr Microbiol Appl Sci. 2015; 4(4): 534–44. Reference Source\n\nMaraki S, Samonis G, Rafailidis PI, et al.: Susceptibility of urinary tract bacteria to fosfomycin. Antimicrobial Agents Chemother. 2009; 53(10): 4508–4510. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcHugh CP, Zhang P, Michalek S, et al.: pH required to kill Enterococcus faecalis in vitro. J Endod. 2004; 30(4): 218–19. PubMed Abstract \| Publisher Full Text\n\nGani BA, Chismirina S, Hayati Z, et al.: The ability of IgY to recognize surface proteins of Streptococcus mutans. Majalah Kedokteran Gigi. 2009; 42(4): 189–93. Publisher Full Text\n\nNematollahi A, Decostere A, Pasmans F, et al.: Adhesion of high and low virulence Flavobacterium psychrophilum strains to isolated gill arches of rainbow trout Oncorhynchus mykiss. Dis Aquat Organ. 2003; 55(2): 101–107. PubMed Abstract \| Publisher Full Text\n\nSasidharan S, Chen Y, Saravanan D, et al.: Extraction, isolation and characterization of bioactive compounds from plants’ extracts. Afr J Tradit Complement Altern Med. 2011; 8(1): 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGamble R, Muriana PM: Microplate fluorescence assay for measurement of the ability of strains of Listeria monocytogenes from meat and meat-processing plants to adhere to abiotic surfaces. App Environt Microbiol. 2007; 73(16): 5235–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBachtiar EW, Bachtiar BM, Dewiyani S, et al.: Enterococcus faecalis with capsule polysaccharides type 2 and biofilm-forming capacity in Indonesians requiring endodontic treatment. J Invest Clinic Dentistry. 2015; 6(3): 197–205. PubMed Abstract \| Publisher Full Text\n\nSoraya CS, Ishaluddin A: Activity antibacterial of propolis on growth of Streptococcus mutans and Enterococcus faecalis based on invitro. Cakradonya Dent J. 2011; 2(3): 332–39.\n\nGani BA, Alghassani AQ, Mubarak Z, et al.: Potensi cigarette smoke condensate terhadap peningkatan pembentukan biofilm Candida albicans isolat ATCC 10261. [The potential of cigarette smoke condensate on increase of biofilm formation of Candida albicans ATCC 10261]. J Syiah Kuala Dent Society. 2017; 2(1): 33–39. Reference Source\n\nSitanggang P, Tambunan E, Wuisan J: Uji kekerasan Komposit terhadap rendaman buah jeruk nipis Citrus aurantifolia. [Hardness assay on composite in lime extract Citrus aurantifolia immersion]. e-Gigi. 2015; 3(1): 229–34. Reference Source\n\nWongkhantee S, Patanapiradej V, Maneenut C, et al.: Effect of acidic food and drinks on surface hardness of enamel, dentine, and tooth-coloured filling materials. J Dent. 2006; 34(3): 214–20. PubMed Abstract \| Publisher Full Text\n\nHugenholtz J: Citrate metabolism in lactic acid bacteria. FEMS Microbiol Rev. 1993; 12(1–3): 165–78. Publisher Full Text\n\nMorandi S, Brasca M, Alfieri P, et al.: Influence of pH and temperature on the growth of Enterococcus faecium and Enterococcus faecalis. Le Lait. 2005; 85(3): 181–92. Publisher Full Text\n\nTypas A, Banzhaf M, Gross CA, et al.: From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Microbiol. 2012; 10(2): 123–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFabretti F, Theilacker C, Baldassarri L, et al.: Alanine esters of enterococcal lipoteichoic acid play a role in biofilm formation and resistance to antimicrobial peptides. Infect and Immun. 2006; 74(7): 4164–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlancato VS, Magni C, Lolkema JS: Functional characterization and Me ion specificity of a Ca-citrate transporter from Enterococcus faecalis. The FEBS J. 2006; 273(22): 5121–30. PubMed Abstract \| Publisher Full Text\n\nSarantinopoulos P, Kalantzopoulos G, Tsakalidou E: Citrate metabolism by Enterococcus faecalis FAIR-E 229. Appl Environ Microbiol. 2001; 67(12): 5482–87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVaroni ME, Lodi G, Sardella A, et al.: Plant polyphenols and oral health: old phytochemicals for new fields. Curr Med Chem. 2012; 19(11): 1706–20. PubMed Abstract \| Publisher Full Text\n\nCotter PD, Hill C: Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiol Mol Biol Rev. 2003; 67(3): 429–453. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoizzo MR, Tundis R, Bonesi M, et al.: Evaluation of Citrus aurantifolia peel and leaves extracts for their chemical composition, antioxidant and anti-cholinesterase activities. J Sci Food Agric. 2012; 92(15): 2960–67. PubMed Abstract \| Publisher Full Text\n\nSandoval-Montemayor NE, Garcia A, Elizondo-Treviño E, et al.: Chemical composition of hexane extract of Citrus aurantifolia and anti-Mycobacterium tuberculosis activity of some of its constituents. Molecules. 2012; 17(9): 11173–84. PubMed Abstract \| Publisher Full Text\n\nMubarak Z, Soraya C: Dataset 1 in: The acid tolerance response and pH adaptation of Enterococcus faecalis in extract of lime Citrus aurantiifolia from Aceh Indonesia. F1000Research. 2018. Data Source\n\nMubarak Z, Soraya C: Dataset 2 in: The acid tolerance response and pH adaptation of Enterococcus faecalis in extract of lime Citrus aurantiifolia from Aceh Indonesia. F1000Research. 2018. Data Source\n\nMubarak Z, Soraya C: Dataset 3 in: The acid tolerance response and pH adaptation of Enterococcus faecalis in extract of lime Citrus aurantiifolia from Aceh Indonesia. F1000Research. 2018. Data Source" }	[ { "id": "31653", "date": "15 Mar 2018", "name": "Boy M. Bachtiar", "expertise": [ "Reviewer Expertise Oral microbiology" ], "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\nThe last paragraph: ………..acid tolerance response and pH adaptation of E. faecalis when it interacts with lime extract.\n\nReviewer’s comment: please change the bold word with: when the bacterium grows as biofilm in the presence of lime extract with difference concentration.\n\nMethod -\n\n“Both materials were made in vitro to analyze”. Reviewer’s comment: please change with: The material and bacterium were prepared\n\n- In lime extract, typo……………………………………into a glass - Culture of E. faecalis bacteria; please discard; “bacteria” - E. faecalis ATCC 29212 taken from glycerol ……………………………………………………………………………………………..and incubator (Memmert, Germany). Reviewer’s suggestion: discard this part, and please replace with: One colony of E. faecalis bacteria was subsequently re-cultured in 5 ml of Mueller-Hinton Broth (MHB) medium (Thermo Fisher Scientific Inc, Paisley, UK) in anaerobic conditions at a temperature of 37°C for 48 hours. Afterward, the E. faecalis grown on the liquid medium was synchronized further with McFarland 0.5 (1 x 108 CFU/ml) (TM50, Dalynn Biological Inc., Calgary, Canada). The Accurate of the density of McFarland standart (typo: it should be standard) can be checked using a spectrophotuometer with an absorbance reading of 0.08 to 0.1 at 625 nm11.\n\nAdaptation to pH assay A total of 50 ml of lime extracts in several different concentrations (100%, 75%, 50%, 25%, 12.5%, and 6.25%) was placed into different beaker glasses. Then, 5 ml containing 1 X 108 CFU/mL of E. faecalis in MHB (1:10) were added to each of the beakers. The initial pH Reviewer’s suggestion: please indicate the initial pH that was measured at the zero h., before incubation. Reviewer’s suggestion: Next, bacterium-containing beaker was put into incubator (37°C) for 6 hours, 12, hours, 24 hours, 48 hours, and 72 hours in an anaerobic atmosphere using Anaerogen TM GasPack (Oxoid, Basing stoke UK), at each of these times, the beakers’ pH was measured using a pH meter (Thermo Fisher Scientific Inc, Paisley, UK). Various changes in pH from 0 hours to the specified time can be used as an indicator of whether E. faecalis has a tolerance response to the acidic environment and can adapt to changing pH.\n\nAcid tolerance assay -\n\n“Here, 96 wells of the triple microplate series were coated with 50 μl of MHB”. Reviewer suggestion: Please change to: Here, 50 ul of MHB was put into microplate in triplicate. -\n\n“the test materials (E. faecalis + lime extract) derived……. Reviewer suggestion: please change to: material tested and E. faecalis……………………………………………”Each well” change to the microplate was put into incubator for 15 min. -\n\n“Then bio-tolerant activity was measured using the Elisa Reader (Bio-Rad Laboratories, Hercules, CA) at a wavelength of 595 nm”. I suggest to discard this part, as it does not has a clear meaning, ….activity was measure by ELISA reader……..\n\nAdhesion assay -\n\n“The standard protein concentration of the E. faecalis and the active component concentration of the lime extract were measured via the Bradford method” It is not clear, why the authors need to analyze (by measuring) the E. faecalis’s proteins (I assume the whole cell proteins). Thus, what does the authors would like to say with \"the standard protein concentration of E. faecalis and the concentration of active component of the lime extract”?. My suggestion is please discard this part.\n\n-\n\n“Spectrophotometry detected the interaction of E. faecalis with lime extract at a wavelength of 595 nm” It is rather confusing. Spectrophotometry was used to detect the interaction between the bacterium and the lime extract. Did you use this method to determine the bacterium growth rate? I suggest to discard this spectrometric method. Otherwise, please focus the methods (adaptation to pH, acid tolerance, and adhesion assay) used only to address your hypothesis; \"that biofilm formation or bacterial adherence ability contributes to E. faecalis survival in biofilm-related environment in the presence of lime extract.\" -\n\nThe principle of incubation time-based interaction activity on the microplate 96 wells series used in this research based on Gamble’s working principle. I think you only tested the bacterial adherence capability, not interaction activity between E. faecalis and……….? -\n\nIt was modified using violet crystalline and safranin staining. First, 96 wells of the triple microplate series were coated with………..\n\nSuggestion: Please change this part: “First, microplate in triplicate wells. ...................Second, 50 uL of E. faecalis in ( ADD IN WHAT MEDIUM USED) ...........Third, 100 uL of different concentration of LE was added and incubated for..........\n\n-\n\n“A total of 100 μL of Lugol solution was added for 1 minute and then washed with PBS. The rest of the cell metabolism that was not bacterial cells was dissolved in 96% alcohol for 20 seconds until the dye completely removed. 50 μL of safranin solution was added for 2 minutes and then washed again with PBS18. The interaction activity between the lime extract and the E. faecalis bacteria in the microplate wells was assessed via an Elisa reader using a spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 595 nm”\n\nSuggestion:\nAs you only tested mono-species biofilm (E. faecalis is a Gram (+) ve bacterium), please add an explanation, why you used both crystal violet and safranin solution?, and you did it similar to the procedure for Gram staining method. Please discard the word: “The interaction activity between the lime extract and the E. faecalis Bacteria”. It would better to say: the bacterial adherence was assessed using crystal violet and measured using ELISA reader with optical density of 595 nm. Observation of adhesion mass. This method is not necessary for this study, as you have used crystal violet (CV) assay to measure biofilm mass. CV provides a good measure of biofilm mass, although it cannot measure biofilm viability. This means, CV stains both bacteria cells and the polymer substance (carbohydrate) as part of the extra cellular matrix.\n\nStatistical analysis\n\n………..standard deviations for each concentration.\n\nReviewer comment: why there are no SD displayed in each graph shown in the figures\n\nResults\n\n-The experiment was conducted in three replicates\n\nReviewer suggestion:\n\nPlease changed: the experiment was performed in triplicate wells This study showed that the presence of lime extracts decreased pH, but reduction of low pH did not have a significant effect on the ability of E. faecalis to adhere and form biofilm, compared to the control (fosfomycin). All result (crystal violet, adaptation to pH, and acid tolerance assays) are shown in Fig .1-3). Interestingly, the tolerance effect was not influenced by exposure time and the concentration of LE set in this study (Fig. 3), and the correlation between time exposure and LE concentration was positive (However, the authors need to show the regression graph as they said r2 = 0.98). Otherwise, please exclude this part. “the results showed that the interaction activities of E. faecalis in Lime extract will decrease from 6, 12, 24, and 72 hours. Reviewer‘s comment: I am wondering what the authors mean with the interaction activity. I suggest to discard this part. The mass profile also indicated interaction activity between lime extract and E. faecalis. Antibiotics are capable of forming a covalent bond to activate the cysteine residue of a bacterial cell, triggering UDP-N-acetylglucosamine to form hydrogen bonds. It inhibits the synthesis of peptidoglycan as an antibacterial defense. Reviewer’s comment: Please discard this part, as 1. I cannot see the data (mass profile). 2. Antibiotic are capable........this is not the result of this study. When necessary please put the sentence in the discussion section.\n\nDiscussion\n\nReviewer’s comment: please focus the results you got. Especially, please do not repeat the result, and exclude: “The interaction activity between lime extract and E. faecalis can be assumed to be the antibacterial activity, because there was a decrease in the interaction activity between E. faecalis and the biological components of lime extract within 6–24 hours and again within 48–72 hours. The interaction activity is related to the activity of the active ingredients contained in the lime extract, such as flavonoids (polyethoxylated flavones and flavanones), coumarin, and terpenoids, all of which act as antibacterials”. I suggest to rewrite this part ……….The survival of E. faecalis in the presence of LE………..\n\nConclusion\n\nE. faecalis can adapt to environments with a pH of 2.9–4.3 generated by lime extracts. In addition E. faecalis also expressed a tolerance response to the acidic environment. The interaction activity between E. faecalis and lime extract become stable within 6–12 hours at a temperature of 37°C. Therefore, the lime extract can be used to inhibit the E. faecalis growth.\n\nReviewer’s comment: Your conclusion should succinctly describe the overall result of the experiment. Please delete: 1. The interaction activity between E. faecalis and lime extract become stable within 6–12 hours at a temperature of 37°C. 2/. Therefore, the lime extract can be used to inhibit the E. faecalis growth.\n\nPlease refer your conclusion to the title that reflects the result.\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? Yes", "responses": [ { "c_id": "3557", "date": "11 Apr 2018", "name": "Zaki Mubarak", "role": "Author Response", "response": "Revision has been made according to the comment of the reviewer" } ] }, { "id": "31610", "date": "19 Mar 2018", "name": "Elza I. Auerkari", "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 general, the paper would still benefit from a review by a native English speaker to correct the language.\nAdditional recommended actions to consider are the following:\nAbstract in Methods (2nd line): instead of “Both materials were analyzed for their adaptation, …” please use “The microbe was analyzed for its adaptation, …”\n\nMethods\nin Culture of E. faecalis bacteria: please rephrase the last sentence “The Accurate of the density ...” to make it more understandable. in Adaptation to pH assay: first sentence: how do the concentration values, given here in %, compare with units of Figures 1 to 3 where concentration is given in micrograms per milliliter?\nResults, Discussion\nFigure 1: why is the initial pH value (at 0 hours) about the same at all lime extract concentrations? Are there any known inhibitor (buffering) substances in the lime extract (or in the bacterial culture) that could explain relatively constant initial pH at a range of concentrations? Figure 2: why is the indicated acid tolerance apparently higher towards increasing lime extract concentration also at 0 hours of exposure? Figure 3: why is the indicated adhesion level apparently higher towards increasing lime extract concentration also at 0 hours of exposure? Where are the error bars mentioned in the text? The paper is referring in many places to “interaction activity” between lime extract and E. faecialis. However, could the results also involve a concentration-dependent interaction between e.g. citric acid and other antibacterial compounds of the extract?\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3556", "date": "11 Apr 2018", "name": "Zaki Mubarak", "role": "Author Response", "response": "We have made the revision based on suggestion and comment from reviewers" } ] } ]	1	https://f1000research.com/articles/7-287
https://f1000research.com/articles/7-447/v1	11 Apr 18	{ "type": "Research Article", "title": "Comparison of hs-CRP level between low calorie high protein to standard protein diet in obese individuals with weight cycling - a randomised trial", "authors": [ "Adventia Natali Paranoan", "Joan Jutamulia", "Septian Ika Prasetya", "Ninik Mudjihartini", "Fiastuti Witjaksono", "Adventia Natali Paranoan", "Joan Jutamulia", "Septian Ika Prasetya", "Ninik Mudjihartini" ], "abstract": "Background: Obesity is associated with various health problems. Low-grade inflammation is a condition found in obese people and contributes to the development of various diseases. The objective of this study was to compare the effects of calorie restriction diet with high-protein (HP) or standard protein (SP) to inflammation marker (hs-CRP) in obese individuals with weight cycling. Methods: 61 healthy obese men and women (25 – 49 years old) with a history of weight cycling were recruited and were randomly assigned to one of the intervention groups, HP or SP groups. Both groups were suggested to reduce their daily caloric intake by 1000 kcal with regular physical activity for 56 days. Subjects in HP group were given a daily protein intake of 22-30% from total daily caloric intake, while SP group were prescribed 12-20%. Dietary consultation was conducted through daily reminder by phone and weekly counseling. The measurement of hs-CRP level was performed prior to and at the end of the intervention. Results: 54 subjects completed the program, yet due to several reasons only 32 of them were measured for hsCRP before and after completing the program, 15 from HP group and 17 from SP group. After completing the 56-day diet program, SP group experienced reduction of hs-CRP by -0.446 + 4.239, while HP underwent increase by 0.135 + 2.389. The mean difference of change in hs-CRP level between the two groups were not statistically significant (P=0.094). Conclusion: Low calorie diet with either HP or SP for 8 weeks significantly reduced body weight (P<0.001) and BMI (P<0.001) in healthy obese subjects but the difference of change in the hs-CRP level between them were not significant. The protein composition of a low calorie diet may not affect the inflammatory state of obese individuals with weight cycling. Trial registration number: NCT03374150", "keywords": [ "high-protein diet", "hs-CRP", "obese", "weight cycling" ], "content": "Introduction\n\nThe global prevalence of obesity according to the World Health Organization (WHO) in 1980 was 5% in the male population and 8% in the female population. In 2014, this increased to 38% in men aged over 18 years and 40% in women. The WHO stated that in the Southeast Asian regions, the prevalence of overweight population varies from 7.6% in male adults in Bangladesh to 53% female adults in Maldives1,2. In Indonesia, a report by RISKESDAS showed that the prevalence of central obesity increased from 18.8% in 2007 to 26.6% in 2013, and the prevalence for males aged >18 years old in the country was 19.7% and females >18 years old was 32.9%. Jakarta province has an obesity prevalence higher than the national rate, 30% for men and 40% for women3.\n\nObesity is associated with various health problems, mainly hypertension, cardiovascular diseases, dyslipidemia, diabetes, sleep apnea, and osteoarthritis. Obese people are more likely to be disabled in their later years, and many obese-related illnesses cause people in productive years to lose their productivity4. Attempts to lose weight by managing dietary intake and increasing physical activity are frequently conducted by many people, but the difficulty in maintaining body weight causes a fluctuation in body weight, called weight cycling5,6. A study by Votruba et al. (2002) reported that 16 (57%) of 28 women regained 19% of the body weight they lost after 1 year of follow-up7.\n\nObesity is accompanied by low-grade inflammation, which contributes to the development of cardiac complications4. The mild increase in hs-CRP levels, as an indicator of low-grade inflammation, is an indepent predictive factor of future cardiovascular diseases8. C-reactive protein (CRP) is an abnormal protein that appears in the blood during inflammation. Highly specific (hs-CRP) test can detect low CRP levels (0,2 mg/L) compared to CRP (N≤5 mg/L)4,9. A previous study by Azadbakht et al. showed that energy-restricted HP and SP diet brought about similar hs-CRP reduction in overweight and obese people8. Further studies are needed to asesss whether in obese individuals with weight cycling history, protein composition could affect inflammation markers. Therefore, the dietary composition that will give the best results for body composition as well as inflammation marker improvement for such population should be investigated. The aim of this study is to evaluate the effects of a low calorie high protein diet compared to a standard protein diet on the level of hs-CRP.\n\n\nMethods\n\nThis is a part of an open-randomized parallel trial assessing the effect of a weight loss program with low calorie high protein diet conducted through dietary consultation on body composition, oxidative stress, inflammation marker and metabolic syndrome in obese individuals with weight cycling. The main interest of this article is the change of hsCRP levels among the HP and SP group before and after completing this program. There are two intervention groups with the allocation ratio 1:1, namely high protein (HP) group and standard protein (SP) group. There were no changes made to the methods after the study had been started (original trial protocol can be found in our sister article10).\n\nApproval from the Health Research Ethical Committee of the Faculty of Medicine Universitas Indonesia – Cipto Mangunkusumo Hospital has been obtained by the letter number 237/UN2.F1/ETIK/2017. Written informed consent for participation in the trial and publication of patient information was obtained from each patient. A completed CONSORT checklist can be found in Supplementary File 1.\n\nTrial registration number: NCT03374150\n\nRegistry name: Clinicaltrials.gov\n\nDate of trial registration: 12/03/2017\n\nTrial link: https://clinicaltrials.gov/show/NCT03374150\n\nThis study was conducted at the province’s Civil Workers’ Health Service Centre of the Special Capital Region of Jakarta (Pusat Pelayanan Kesehatan Pegawai Provinsi DKI Jakarta). Initial screening criteria for subject inclusion was men and women age > 20 years old with body mass index ranged 25 – 35 kg/m2. In this study, weight cycling was defined by history of weight loss ≥2 kg and regaining weight into or exceeding its initial body weight at least twice in last five years. Weight cycling history was obtained from a questionnaire regarding history of body weight changes (see supplementary materials of our sister article10). Subjects were excluded if they had diabetes mellitus, history of gastrointestinal tract resection, hormonal disorders, using hormonal contraception, menopause, and having kidney function disruption, which was identified from serum urea and creatinine levels. The recruitment and intervention were conducted in two periods, namely May – July and July – September of 2017.\n\nAll subjects who meet the inclusion criteria were measured for anthropometric data and were instructed to cease their previous diet program and requested to continue performing their daily physical activity at a regular level. History of previous daily calorie intake was gained from food recall interview 24 hours by asking type, cooking method, and estimation of amount of food consumed using household size, based on food photo books issued by Tim Survey Konsumsi Makanan Individu, Ministry of Health of Indonesia.\n\nDietary consultation for weight loss program was given for each subject in which all of them were suggested to reduce their daily caloric intake by 1000 kcal from their regular amount with lower limit of 1000 kcal/day. Subjects of HP group were assigned to low calorie-high protein diet with a composition of 22–30% protein, 50–55% carbohydrate, 20–25% fat while SP group were low calorie-balanced composition diet with 12–20% protein, 55–60% carbohydrate, and 20–30% fat. In order to ensure the compliance, follow-up to each subject were conducted by weekly person-to-person encounter with nutritionist and daily text reminder Subjects were equipped with diet formula, and logbook. Dietary program was applied for 8 weeks and subjects were instructed to fill logbook everyday to record their food intake. The measurement for hs-CRP level was done pre-intervention and re-measured after each subject has completed the 8 weeks of diet intervention.\n\nA total of 28 participants would be needed in order that the difference between groups could be detected with a two-tailed α of 0.05 and a (1-β) of 0.80. Subject allocation into the intervention arms was determined through block randomization method with a block size of two. The group allocation sequence was constructed by the investigator (JJ) by random number generation method in which a computer-generated random number was taken, even number represent an arrangement of HP-SP and vice versa. The allocation sequence for all the participants that will be used throughout the study was generated by the investigator before the study commenced. Envelopes each representing a particular group were arranged according to the allocation sequence and then were orderly numbered. It was given consecutively to each subject who come hence the allocation sequence was blinded from the subject. The investigator assigned the subjects to the treatment group according to the envelope he/she received.\n\nThe primary data of this study is serum hs-CRP level before and after the diet intervention, which was measured using Immunochemmiluminescent device (Immulite 1000 Immunoassay system, Siemens Healthineers, Erlangen, Germany). The secondary outcome was dietary profile comprising mean daily caloric intake and mean daily proportion of macronutrients during the 56-days of diet program. Dietary data was measured with 24 hours food recall, while subject’s physical activity was calculated using Global Physical Activity Questionnaire from the WHO. Body weight and BMI measurement were conducted using 8-electrode method of Bio-Electrical Impedance Analysis (BIA; SC-330 Tanita Corp. Tokyo, Japan). No change to the outcome measures was done after the study commenced.\n\nThe characteristics of subjects were analyzed statistically using independent sample t-test (age and previous calorie intake), Fisher’s exact test (gender and weight cycling history), and Mann-Whitney test (weight and Body Mass Index (BMI)). All data except for body weight in HP group given as mean wih 95% confidence interval. Thirty-six subjecs were included for analysis. Change in body weight and BMI analyzed statistically using independent sample t-test, while intragroup pre- and post- mean difference of body weight and BMI analyzed by Wilcoxon test. The value of measured hs-CRP analyzed statistically by Mann-Whitney test. Statistical analysis was performed using SPSS for Windows version 20.\n\n\nResults\n\nThe number of participants who were screened, randomised and allocated into treatment, completed the treatment and analysed are provided in Figure 1. There were 54 participants completed the 56-days of diet program but only 15 subjects from SP group and 17 subjects from HP group who completed the treatment and had their hsCRP been measured were analyzed for the primary outcome to the group they were initally assigned. During the course of treatment, no major harms were reported by the participants.\n\nCharacteristics of subjects are presented in Table 1. There is no statistically significant difference found in the subjects’ characteristics. Meanwhile, dietary profile of the subjects among both groups during the 56-days of dietary programme were presented in the Table 2. Subjects’ body weight and BMI before and after the programme as well as the difference are provided in Table 3. The body weight, BMI, as well as the change in the body weight and BMI before and after the diet programme were higher in the SP group than the HP group, although those were not statistically significant. SP and HP groups both showed significant intragroup mean difference in body weight and BMI reduction pre- and post intervention. SP group experienced slightly greater reduction in body weight and BMI compared to HP protein (body weight 0.216, BMI p=0.136). The hs-CRP level before and after completing the intervention are provided in the Table 4. The hs-CRP level before (P=0.094) and after (P=0.063) the diet programme were higher in the SP group than in the HP group. After completing the 56-day diet programme, SP group experienced reduction of hs-CRP by -0.446 ± 4.239 while HP underwent overall increase by 0.135 ± 2.389 (p=0.094).\n\nOnly subjects who completed the study were included.\n\nValues are expressed as mean ± SD except avalues are medians (min- max)\n\nThere were no differences between groups (P>0.05) by t Independent samples t-test, mMann-Whitney test, fFisher’s exact test\n\nBMI = Body Mass Index\n\nsignificance was set at <0.05\n\ntIndependent samples t-test\n\nmMann-Whitney test\n\nCompliance to the daily diet plan of 1000-kcal caloric intake restriction and proportion of caloric intake of 12–20 per cent for the SP group or 22–30 per cent for the HP group\n\nsignificance was set at <0.05\n\ntIndependent samples t-test\n\nmMann-Whitney test\n\npPaired-samples t-test\n\nwWilcoxon test\n\nsignificance was set at P<0.05\n\nmMann-Whitney test\n\nwWilcoxon test\n\n\nDiscussion\n\nThe baseline and post-treatment hs-CRP level of HP group was lower than that of the SP group, although both were insignificant. The change of hs-CRP level of both group were also statistically insignificant. While the mean hs-CRP level of HP group increased, in the SP group it decreased. The insignificant reduction in hs-CRP despite weight loss and the decrease of BMI may because all the subjects were still in the overweight or obese state. Adipokines released by adypocites or macrophages induced low-grade chronic inflammation in overweight and obese people11; hence the hs-CRP level as the indicator of such condition is maintained.\n\nThe result of this study is similar to a study by Due et al., in which SP diet in overweight subjects showed a better reduction of hs-CRP than HP diet12. The result is also in line with a study by Azadbakht et al. on the effects of HP diet to weight, cardiovascular risk and hs-CRP. Between the HP-low calorie diet (protein, carbohydrate, and fat: 25%, 45%, and 30% respectively) and SP-low calorie diet (protein, carbohydrate and fat : 15%, 55%, and 30%), hs-CRP in the standard group decreased greater by which it reduced −0.08 ± 0.11%, while in the HP group it only reduced by −0.04 ± 0.09% (p=0.06)13. Another study by Kitabchi et al., which compared various metabolic marker in the HP diet and high carbohydrate diet for 6 months showed a greater decrease of inflammation marker (CRP) in the group receiving high carbohydrate diet than in the HP group (-2,1 vs. -0.8 mg/L, p=0.0003)14.\n\nDespite the benefit of higher protein diet in increasing satiety, the present study found that SP composition provided larger reduction in body weight and hs-CRP level compared to HP group. The amino acids obtained from protein intake were absorbed then are passed into the liver. The rate of synthesis and degradation of amino acids there is influenced by many factors including immediate food intake and nutritional status14. Inflammation and physical inactivity in the weight cycling condition which were characterized by accelerated wasting of lean body mass lead to the accelerated depletion of amino acids obtained from the dietary protein intake to counter the amino acids loss during the degradation of protein from body mass15.\n\nA major limitation of this study is the absence of detailed assessment of the condition of the participants during the course of the intervention, such as the presence of acute illness, the use of drugs that affect the body inflammatory state and so on, which potentially affect the level of hs-CRP. Besides, the dietary consultation failed to maintain the compliance of the participants to the dietary plan to the desired extent. The average number of days in which subjects comply towards the assigned program is only 20 days in the SP group and 26 in the HP group which may further limit the validity of this study. The majority of participants of this study were office workers which may limit the generalisability of this study.\n\n\nConclusions\n\nAlthough both standard and high protein low-calorie diet for 56 days reduced body weight and BMI significantly in obese people with weight cycling, SP diet of 12–20% of overall daily caloric intake decreased hs-CRP levels, as an inflammation marker, greater than HP diet. However, the mean difference of change in hs-CRP level between the two groups were not statistically significant. This is probably due to the subjects from both groups were still in the obese/overweight state hence the change in the level of inflammation marker were less prominent. A trial with larger participants and/or longer follow-up period are needed to confirm whether protein proportion of dietary programme affects the change in serum hs-CRP level.\n\n\nData availability\n\nDataset 1: Harvard Dataverse. Replication Data for Comparison of low calorie-standard protein or high protein diet on body composition, malondialdehyde and glutathione, and hs-CRP level, http://dx.doi.org/10.7910/DVN/7H55FP16\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\nPublikasi Internasional Terindeks Untuk Tugas Akhir Mahasiswa (PITTA) grant 2017, Directorate of Research and Community Services (DRPM) Universitas Indonesia.\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: CONSORT checklist.\n\nClick here to access the data.\n\n\nReferences\n\nWHO: WHO \| Obesity and overweight. World Heal Organ Media Cent Fact Sheet No 311 [Internet]. 2012; 1–2. Reference Source\n\nWHO: WHO _ Obesity and overweight [Internet]. [cited 2018 Dec 1]. Reference Source\n\nBadan Penelitian dan Pengembangan Kesehatan: Riset Kesehatan Dasar (RISKESDAS) 2013. Lap Nas 2013. 2013; 1–384. Reference Source\n\nPoirier P, Giles DG, Bray GA, et al.: Obesity and cardiovascular disease: pathophysiology, evaluation, and effect of weight loss: an update of the 1997 American Heart Association Scientific Statement on Obesity and Heart Disease from the Obesity Committee of the Council on Nutrition, Physical Activity, and Metabolism. Circulation. 2006; 113(6): 898–918. PubMed Abstract \| Publisher Full Text\n\nPolsky S, Catenacci V, Wyatt HR: Obesity: Epidemiology, Etiolgy and Prevention. In: Ross C, Caballero B, Cousin RJ, Tucker CL, editors. Modern Nutrition in Health and Disease. 11th ed. Philadephia: Lippincott Williams & Wilkins; 2014; 772–86.\n\nStrohacker K, Carpenter K, McFarlin BK: Consequences of Weight Cycling: An Increase in Disease Risk? Int J Exerc Sci. 2009; 2(2): 191–201. PubMed Abstract \| Free Full Text\n\nVotruba SB, Blanc S, Schoeller DA: Pattern and cost of weight gain in previously obese women. Am J Physiol Endocrinol Metab. 2002; 282(4): E923–30. PubMed Abstract \| Publisher Full Text\n\nSellmayer A, Limmert T, Hoffmann U: High sensitivity C-reactive protein in cardiovascular risk assessment. CRP mania or useful screening? Int Angiol. 2003; 22(1): 15–23. PubMed Abstract\n\nBrunner S: Handbook of Laboratory and Diagnostic Tests. Philadephia: Lippincott Williams & Wilkins; 2010; 200–201.\n\nJutamulia J, Paranoan AN, Prasetya SI, et al.: Comparison of body composition changes between low calorie high protein diet to standard protein in obese individuals with weight cycling – a randomised trial. F1000Research. Publisher Full Text\n\nLeal Vde O, Mafra D: Adipokines in obesity. Clin Chim Acta. 2013; 419: 87–94. PubMed Abstract \| Publisher Full Text\n\nDue A, Toubro S, Stender S, et al.: The effect of diets high in protein or carbohydrate on inflammatory markers in overweight subjects. Diabetes Obes Metab. 2005; 7(3): 223–9. PubMed Abstract \| Publisher Full Text\n\nAzadbakht L, Izadi V, Surkan PJ, et al.: Effect of a High Protein Weight Loss Diet on Weight, High-Sensitivity C-Reactive Protein, and Cardiovascular Risk among Overweight and Obese Women: A Parallel Clinical Trial. Int J Endocrinol. 2013; 2013: 971724. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKitabchi AE, McDaniel KA, Wan JY, et al.: Effects of high-protein versus high-carbohydrate diets on markers of β-cell function, oxidative stress, lipid peroxidation, proinflammatory cytokines, and adipokines in obese, premenopausal women without diabetes: a randomized controlled trial. Diabetes Care. 2013; 36(7): 1919–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGroff J, Smith JL: Advanced Nutrition and Human Metabolism. 5th ed. Belmont; 2009; 236.\n\nIka Prasetya S: “Replication Data for Comparison of low calorie-standard protein or high protein diet on body composition,malondialdehyde and glutathione, and hs-CRP level”. Harvard Dataverse, V1, UNF:6:KCV9N2pSzHTzk2CX+6eWQA==. 2018. Data Source" }	[ { "id": "35974", "date": "21 Dec 2018", "name": "Denise Mafra", "expertise": [ "Reviewer Expertise Renal nutrition" ], "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 is confused and the authors did not explain exactly what the hypothesis is? The conclusion in the abstract shows p values, this is not common and not interesting.\nThe authors should write more about the role of protein in inflammation.\nBMI and weight are not body composition measurements.\nThe diets were low carb and low fat and what was the exact protein intake expressed in g/kg/d, because 21% of protein is a diet with high protein intake? Normally, CRP values are not parametric data.\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? 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? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/7-447
https://f1000research.com/articles/7-444/v1	11 Apr 18	{ "type": "Case Report", "title": "Case Report: Suspicions of metastasis in a patient with transitional cell carcinoma were revealed to be spinal tuberculosis", "authors": [ "Owrang Eilami", "Shahla Jahanbin", "Gordafarin Nikbakht", "Faezeh Azarifar", "Saeid Jokar", "Owrang Eilami", "Shahla Jahanbin", "Gordafarin Nikbakht", "Faezeh Azarifar" ], "abstract": "Background: Infection with Mycobacterium tuberculosis (TB) is one of the major causes of mortality in developing countries. TB is primarily a lung disease, but can affect almost every organ of the body. Skeletal TB involves the bones or joints. In this report, we will introduce a patient with a medical history of transitional cell carcinoma (TCC) of the bladder that presented with spinal tuberculosis (Pott's disease). Case Report: The patient was a 74-year-old man with medical history of TCC of the bladder who had come to hospital due to severe weakness and sprains of lower extremities. Other symptoms noted by the patient included anorexia, weight loss (of 5 kg), and night sweats, but he did not complain of fever, coughs or respiratory symptoms. The lab data were as follows: WBC, 16/9*103; ESR, 88 mm/hr; CRP, 78mg/dl. Radiology findings revealed degenerative process in the L2-L3 lumbar vertebrae and disk. PCR and sample tissue culture results showed the presence of Mycobacterium tuberculosis. Conclusion: In the lesions of the lumbar vertebrae, even if there is another underlying disease, spinal TB should also be considered as a possibility. Furthermore, in patients with any type of cancer, any other organ conflict is not considered as metastasis, and tissue sampling should be provided because a change in the type of disease can influence prognosis.", "keywords": [ "Mycobacterium Tuberculosis", "Spondylitis", "Potts disease", "metastasis", "Transitional Cell Carcinoma" ], "content": "Introduction\n\nInfection with Mycobacterium tuberculosis (TB) is one of the major causes of mortality in developing countries, affecting millions throughout the world1,2. TB is primarily a lung disease but can affect almost every organ of the body. The term \"extrapulmonary TB\" is used to describe a clogged infection in places other than the lung. The most common places are extrapulmonary tuberculosis of the lymph nodes, urinary tract, pleura, bones and joints, meninges and central nervous system, peritonea and other abdominal organs3. In a study of 483 patients with pulmonary TB infection in Chile, only 2% of all the cases of tuberculosis infection were associated with skeletal tuberculosis4. In addition, in the United States, an estimated 10.8% of extra-pulmonary tuberculosis cases were considered skeletal tuberculosis in general, accounting for 2.3% of total tuberculosis statistics5. Spinal TB, also known as “Pott's disease,” accounts for about 50% of cases of skeletal tuberculosis, and is commonly found in children and adolescents6.\n\nIn this report, we will introduce a patient with a medical history of transitional cell carcinoma (TCC) of the bladder that presented with spinal tuberculosis (Pott's disease).\n\n\nCase report\n\nThe patient was a 74-year-old man from Yasouj city (Southwest of Iran) with a medical history of chronic kidney disease, TCC of the bladder, who was on BCG (Bacille Calmette-Guérin) chemotherapy, and deep vein thrombosis, who had come to hospital due to severe weakness and sprains of lower extremities. The patient noted that the weakness and numbness of the lower extremities were progressive and became worse at night. During this period, the patient had not undergone any further diagnosis, and controlled his pain with acetaminophen. Other symptoms that the patient noted was anorexia, weight loss of 5 kg, and night sweats, but he did not complain of fever, cough and respiratory symptoms.\n\nDuring the clinical examination, tenderness of the lumbar spine was accompanied by a decrease in the range of motion (ROM) from 2 / 5 of right lower extremities and 3 / 5 of right lower extremities, in addition to positive reverse SLR (Straight Leg Raise) test.\n\nThe patient’s test results are presented in Table 1.\n\nIn the CT scan, hypo-dense mass of size 140 × 44 × 44 mm in paravertebral space L2 was observed, with destruction of the right and left facet joint and spinous process of L2, and destruction of intervertebral disk of the L2 - L3 (Figure 1). In the MRI, an increase in the signal of the L2 and L3 vertebral bodies was observed, along with the destruction of the anterior plate and the reduction of the articular space. In the same area, a lesion was observed with a moderate signal on the anterior longitudinal ligament and posterior longitudinal ligament, and a complete loss of CSF (Figure 2).\n\nIn the same area, complete loss of CSF can be seen.\n\nFor accurate diagnosis, the patient underwent ultrasound-guided biopsy, and the samples were sent to the lab for PCR, culture and histological examination. In the sampling report, PCR confirmed infection with Mycobacterium tuberculosis. Furthermore, the tissue culture was also found to be positive for Mycobacterium tuberculosis.\n\nAfter diagnosis, treatment was started with isoniazid (300mg daily), rifampin (600mg daily), ethambutol (1.2 gr daily), and pyrazinamide (1.5 gr daily) for 2 months then isoniazid and rifampin for 10 months.\n\nCurrently, after 4 months, the patient receives anti-TB drugs under the supervision of the Yasouj Health Center, and has not noted any evidences of weakness or night sweating. ROM of both lower extremities is 4/5. After completion of treatment, the patient will undergo a follow-up period under the supervision of the Neurosurgery Department.\n\n\nDiscussion\n\nSkeletal TB refers to the involvement of the bones or joints7. Forms of skeletal TB include osteomyelitis, spondylitis, and arthritis. The literature on spinal TB shows a wide variation in reported rates of active concomitant pulmonary TB at the time of spinal TB diagnosis8–10. In our case, however, pulmonary involvement was absent.\n\nTB spondylitis or Pott’s disease most commonly affects the lower thoracic and upper lumbar vertebras, and less frequently cervical and upper thoracic vertebrae10,11. The most common symptom is focal pain, which increases in severity over time, and is sometimes accompanied by muscle spasm. The muscle spasm can extend to other parts of the body. In some cases, it can cause difficulty in gait.12.\n\nThe diagnosis of skeletal TB is often delayed and may be difficult. It is made based on culture of tissue13. But computerized tomography, magnetic resonance imaging, and myelography are all useful diagnostic tools10,14–16. Radiographic findings can be nonspecific; early features may include soft tissue swelling (especially of the anterior portions of the vertebral body) with bone demineralization and preservation of joint surfaces11. In our case, because of the seriousness of decreased range of motion of lower extremities, and high clinical susceptibility to Mycobacterium infection, and given that radiological findings were similar to those for patients with TB spondylitis, the process of diagnosis was rapid.\n\nPatients with metastatic TCC of bladder in the bone and liver have poor prognosis17. For this reason, it was important to rule out metastasis in the case of this patient.\n\nGiven that vertebrae osteomyelitis has been seen in patients receiving intravesical BCG for the treatment of TCC of the bladder18, the presence of Mycobacterium bovis was expected in the culture sample, but Mycobacterium tuberculosis was confirmed.\n\n\nConclusion\n\nIn the lesions of the lumbar vertebrae, even if there is another underlying disease, spinal TB should also be considered as a possibility.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of the patient’s clinical details and accompanying images.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "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\nDye C, Scheele S, Dolin P, et al.: Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA. 1999; 282(7): 677–686. PubMed Abstract \| Publisher Full Text\n\nMoon MS, Moon YW, Moon JL, et al.: Conservative treatment of tuberculosis of the lumbar and lumbosacral spine. Clin Orthop Relat Res. 2002; (398): 40–49. PubMed Abstract \| Publisher Full Text\n\nSandgren A, Hollo V, Huitric E, et al.: Epidemiology of tuberculosis in the EU/EEA in 2010: monitoring the progress towards tuberculosis elimination. Euro Surveill. 2012; 17(12): pii: 20124. PubMed Abstract\n\nArriaza BT, Salo W, Aufderheide AC, et al.: Pre-Columbian tuberculosis in northern Chile: molecular and skeletal evidence. Am J Phys Anthropol. 1995; 98(1): 37–45. PubMed Abstract \| Publisher Full Text\n\nPeto HM, Pratt RH, Harrington TA, et al.: Epidemiology of extrapulmonary tuberculosis in the United States, 1993–2006. Clin Infect Dis. 2009; 49(9): 1350–7. PubMed Abstract \| Publisher Full Text\n\nGarg RK, Somvanshi DS: Spinal tuberculosis: a review. J Spinal Cord Med. 2011; 34(5): 440–454. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaniel TM, Bates JH, Downes KA: History of tuberculosis. In: Tuberculosis: Pathogenesis, Protection, and Control. Bloom BR (Ed), American Society for Microbiology, Washington. 1994. Publisher Full Text\n\nHershkovitz I, Donoghue HD, Minnikin DE, et al.: Detection and molecular characterization of 9,000-year-old Mycobacterium tuberculosis from a Neolithic settlement in the Eastern Mediterranean. PLoS One. 2008; 3(10): e3426. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPertuiset E, Beaudreuil J, Lioté F, et al.: Spinal tuberculosis in adults. A study of 103 cases in a developed country, 1980–1994. Medicine (Baltimore). 1999; 78(5): 309–20. PubMed Abstract \| Publisher Full Text\n\nTurgut M: Spinal tuberculosis (Pott's disease): its clinical presentation, surgical management, and outcome. A survey study on 694 patients. Neurosurg Rev. 2001; 24(1): 8–13. PubMed Abstract \| Publisher Full Text\n\nWeaver P, Lifeso RM: The radiological diagnosis of tuberculosis of the adult spine. Skeletal Radiol. 1984; 12(3): 178–86. PubMed Abstract \| Publisher Full Text\n\nLifeso RM, Weaver P, Harder EH: Tuberculous spondylitis in adults. J Bone Joint Surg Am. 1985; 67(9): 1405–13. PubMed Abstract\n\nVersfeld GA, Solomon A: A diagnostic approach to tuberculosis of bones and joints. J Bone Joint Surg Br. 1982; 64(4): 446–9. PubMed Abstract \| Publisher Full Text\n\nColmenero JD, Ruiz-Mesa JD, Sanjuan-Jimenez R, et al.: Establishing the diagnosis of tuberculous vertebral osteomyelitis. Eur Spine J. 2013; 22 Suppl 4: 579–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShanley DJ: Tuberculosis of the spine: imaging features. AJR Am J Roentgenol. 1995; 164(3): 659–64. PubMed Abstract \| Publisher Full Text\n\nDesai SS: Early diagnosis of spinal tuberculosis by MRI. J Bone Joint Surg Br. 1994; 76(6): 863–9. PubMed Abstract \| Publisher Full Text\n\nLoehrer PJ Sr, Einhorn LH, Elson PJ, et al.: A randomized comparison of cisplatin alone or in combination with methotrexate, vinblastine, and doxorubicin in patients with metastatic urothelial carcinoma: a cooperative group study. J Clin Oncol. 1992; 10(7): 1066–73. PubMed Abstract \| Publisher Full Text\n\nMiyazaki M, Yoshiiwa T, Ishihara T, et al.: Tuberculous Spondylitis following Intravesical Bacillus Calmette-Guerin for Bladder Cancer. Case Rep Orthop. 2016; 2016: 6741284. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "33631", "date": "11 May 2018", "name": "Mohsen Moghadami", "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\nWhat is the result of PPD skin test or Quantiferon assay of the patient?\n\nThe author must determine the exact method of Diagnostic PCR and the type of primer. Many types of MTB PCR exist around the world with variable sensitivity and specificity.\n\nThe author must determine the exact method and type of MTB culture.\n\nRewriting of case presentation with more detail about examination and history and correction of English writing errors by a native English editor.\n\nNeed for the chest x-ray of the patient.\n\nThe author has noted the sample was sent for histopathology. What was the result? The figure of histopathology with specialized staining should be added.\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? Partly", "responses": [] }, { "id": "33125", "date": "13 Jun 2018", "name": "Dilip Singh", "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 is the report describing a patient with Pott's spine who also had cancer of bladder. Patient presented with a picture of compressive myelopathy along with constitutional symptoms. Authors did the typical work up and confirmed the diagnosis of Pott's spine based on microbiological data.\n\nIn the case description, duration and progression of symptoms are not clear.\n\nIn the endemic areas with TB it is not uncommon for patients to present with extra pulmonary TB. Given the typical presentation described in this case, Pott's spine still remains an important differential, despite the known diagnosis of cancer.\n\nIt is well known to scientific community to consider the diagnosis of Pott's spine in such cases in endemic areas hence this case doesn't add anything in the existing literature.\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? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/7-444
https://f1000research.com/articles/7-443/v1	11 Apr 18	{ "type": "Research Article", "title": "Internal transcribed spacer for identification of yeast species isolated from cancer patients at the Isotope and Radiation Center, Khartoum, Sudan: A cross-sectional, case-control study", "authors": [ "Mohamed M.A. Nagla", "Omer E. El Fadil", "Abdel Hamid M. Muzamil", "Altayeb N. Hisham", "Mohamed B. Bahaeldeen", "El-Amin El-Nour", "Omer E. El Fadil", "Abdel Hamid M. Muzamil", "Altayeb N. Hisham", "Mohamed B. Bahaeldeen", "El-Amin El-Nour" ], "abstract": "Background: Cancer patients have a high risk of fungal infections, especially by Candida species. Non-C. albicans Candida infections and less common yeast infections have been increasing in recent years. Identification by conventional methods can be difficult and sometimes inconclusive. This study aimed to detect the prevalence of oral yeast species isolated from cancer patients, from oral swab, sputum and urine, using Internal Transcribed Spacer (ITS) sequence analysis, since little is known about this problem in Sudan. Methods: The study involved 333 cancer patients (168 patients under treatment [study group] and 165 patients before treatment [control group]). Oral swabs were collected from all patients. Urine or sputum specimens were collected from patients under treatment showing clinical features of UTI or lower respiratory tract infection, respectively. ITS1 and ITS2 region of isolated yeast were amplified by PCR and sequenced. The obtained sequences were compared to reference sequence available in the GenBank database using BLAST. Results: Culture results showed oral yeast species were isolated from 69/168 (41.1%) and 74/165 (44.8%) of patients among study and control groups, respectively (P value > 0.05). 2/9 (22.2%) patients were urine growth positive and 8/14 (57.1%) patients were sputum culture positive. Sequence analysis showed, C. albicans was the most prevalent organism (93; 52.5%) followed by C. tropicalis (29; 16.4%), and C. glabrata (24; 13.6%). Non-C. albicans Candida and uncommon rare yeast were found to be associated with oral infections and colonization among the study and control groups, whereas C.albicans was the most common species (66.7%) associated with oral candidiasis among the treated patients.\n\nConclusion: Cancer patients were highly colonized with different oral yeast species, which indicates that ITS sequence analysis is an accurate method for identification. This will aid effective management to prevent dissemination of disease especially among those who are under chemo and/or radiotherapy treatment.", "keywords": [ "Yeast species", "ITS sequencing", "Cancer patients." ], "content": "Introduction\n\nAs a result of the immunocompromised state and the effect of chemotherapy, cancer patients are more susceptible to fungal infections, especially by Candida species (AI-Dwairi et al., 2014). Candida species are commensal in human bodies, and become opportunistic pathogens in immunodefective patients. Sometimes they can cause systemic infections and colonize different organs due to their dissemination from the mucosal infected regions (Miceli et al., 2011). Pfaller et al. (2006) and Wilson et al., (2002) reported that the systemic infections have considerable morbidity among those with severely paralyzed immune system.\n\nThe most common fungal infection is oropharyngeal candidiasis, which is more prevalent among cancer patients (Akapan & Morgan, 2002). Recently infections with non-Candida albicans and rare common yeast genera, such as Pichia, Rhodotorula, and Saccharomyces, have been implicated; however, C. albicans remains the most prevalent species (Han et al., 2004 and Walsh et al., 2004).\n\nYeast identification is of great importance for targeting proper treatments (Ramani et al., 1998), since different species yield different antifungal response (Pfaller et al., 2003). Conventional methods for yeast identification may be difficult and inconclusive (Reiss et al., 1998) especially for less common yeast. Thus more rapid and accurate molecular methods have been developed, among which ITS sequence analysis is found to be more accurate for species delineation (Chen et al., 2001 and Iwen et al., 2002).\n\nThe ITS region is located between the highly conserved genes coding for 18S and 28S rRNA. The ITS region includes two none coding regions ITS1 and ITS2, which are separated by the highly conserved 5.8SrRNA gene (White et al., 1990). The more genetic variability of ITS1 and ITS2 regions enables better identification of closely related species other than the adjacent rRNA gene (Ciardo et al., 2006).\n\nThis study aimed to detect the prevalence of oral yeast species isolated from cancer patients by oral swab, in addition to other specimens (sputum and urine), using ITS sequence analysis, since little is known about this problem in Sudan.\n\n\nMethods\n\nThis was a cross-sectional, case-control study conducted in a period between April 2013 and December 2017.\n\nThe study involved 333 cancer patients referred to Isotope and Radiation Center, Khartoum, who were seeking anticancer treatment and during a routine check-up were enrolled in this study. The participants were classified into 168 patients under chemo and/or radiotherapy treatment (study group) and 165 cancer patients prior to starting anticancer treatment (control group). Study participants included 185 females and 148 males (mean age, 48 years old).\n\nWritten informed consent and structural questionnaire (see Supplementary File 1), including demographical data, site of cancer and cancer treatments, was obtained from each patient. The participants were ensured of anonymity and that only group findings will be reported.\n\nTo reduce any possible bias matching criteria was done, which included age, sex and type of cancer. Inclusion criteria: Any patient diagnosed with any type of cancer during or before starting cancer treatment, having age equal or above 18 year old, attending Isotope and Radiation Center, Khartoum was included in this study. Exclusion criteria: Patients on antifungal therapy for past two weeks were excluded from the study.\n\nOral swabs were collected from each patient (333 patients) to detect the prevalence of yeast infections and colonization among the treated and non-treated patients. Urine (n=9) and sputum (n=14) samples were collected from patients under treatment (study group), who exhibited the clinical features of urinary tract infection (UTI) and/or lower respiratory tract infection (LRT), respectively. These samples were taken in order to detect whether there is any dissemination from a patient’s own oral yeast due to action of cancer treatments.\n\nAll specimens (n=356) were cultured without delay in Sabouraud’s dextrose agar plates (SDA) to which chloramphenicol (0.05g/l) was added. Then the plates were incubated at 37°C for 24–48 hours. Phenotypic identification was made using Gramʹs stain and germ tube test. Purified colonies were preserved on glycerol stock solution for molecular identification (Sherman et al., 1986).\n\nThe isolated strains were subbed from the stock solution on Sabouraud’s dextrose agar medium and DNA extraction was performed from colonies that had been incubated for 48 hrs using Guanidine Chloride method as described by Gassoum et al. (2014). Three to five colonies were washed with 5 ml phosphate buffer saline (PBS) (Sigma Aldrich) for three times. 2 ml white cell lysis buffer and 20 µl of proteinase K (10 mg/ml; iNtRON Inc, Korea) were added, vortexed and incubated at 37°C for overnight. Then 1 ml from Guanidine chloride (7M; iNtRON Inc, Korea) and 350 µl of ammonium acetate (7M; Loba Chemie, India) were added. The tubes were vortexed and incubated at 65°C in an oven for 2 hours. Then the supernatant was mixed with 2ml pre chilled chloroform (sd Fine-Chem limited, India) at 6000 RPM for 20 minutes and this was transferred into a new Falcon tube and completed to 10 ml volume with pre chilled absolute ethanol (Carlo Erba, France) and incubated overnight at -20°C for completion of DNA precipitation.\n\nAfter incubation the tubes were centrifuged at 6000 RPM for 20 minutes, then the ethanol was poured off and the same step was repeated with 70% ethanol. After that the tubes were left to air dry. Finally DNA was suspended in 80 µl TE buffer (iNtRON Inc, Korea) and incubated at 4 °C until used. Nanodrop ND 1000 Spectrophotometer (NanoDrop Technologies, Inc.) was used to measure quality and quantity of DNA.\n\nThe universal fungal primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) (Macrogen Inc. Korea) were used to amplify the entire ITS rDNA region (Zimbeck et al., 2010). PCR mixture contained 5 μl pre mix (iNtRON Inc., Korea), 22μl deionized sterile water, 1 μl from each forward and reverse primer, and 1 μl of genomic DNA, which served as the DNA template in a final volume of 25 μl.\n\nPCR cycling conditions were as follows: an initial denaturation step of 5 min at 95°C followed by 35 cycles of 45 s at 94°C, 45 s at 55°C, and 45 s at 72°C, with a final extension of 5 min at 72°C. The reactions were carried out in an ESCO thermocycler (AERIS-BG096, China).\n\nThe PCR products were analyzed on 2% agarose gels (iNtRON Inc., Korea) stained with ethidium bromide (10 ng/100 ml; Fisher Scientific, USA) and visualized under a UV transilluminator apparatus (Saratoga, CA.95070, USA Gel Documentation System) and Biodoct BDA system (Biometra, Germany).\n\nPCR products of 90 isolates from the cases and 87 isolates from the control group were purified and commercially sequenced using forward primer ITS1 and backward primer ITS4 by Macrogen Company (Seoul, Korea).\n\nThe sequences obtained in this study were identified by searching databases using BLAST sequence analysis tool (http://www.ncbi nlm.nih.gov/BLAST/). The sequences were compared using nucleotide-nucleotide BLAST (blastn) with default setting except that sequences were not filtered for low complexity. Species were identified based on the highest similarity score (100%) with reference database sequence.\n\nData was analyzed using SPSS 21. Frequencies and percent were obtained for frequency tables, Chi-squared test was used for goodness of fit. The relationships between variables tested were obtained using cross tables and Chi-squared (Fisher exact) test for independence. P-value ≤0.05 was considered as significant.\n\nScientific approval was obtained from Faculty and Department of Medical Microbiology Management, Al-Neelain University (No AU/FMLS/7/1). Ethical clearance was obtained from the Directorate of Research Department, Ministry of Health, Khartoum (dated on 21-04-2013).\n\n\nResults\n\nThe collected oral swabs (333 specimens), from 168 cancer patients with chemo and/or radiotherapy treatment (study group) and from 165 cancer patients (control group) without treatment were examined by cultural technique. Oral Candida species were isolated from 69/168 (41.1%) and 74/165 (44.8%) of patients among study and control groups, respectively.\n\nOf the 168 study group, 23 patients with clinical symptoms of UTI and/or LRT infection had urine (n=9) and sputum (n=14) samples collected for detection of Candida species. The results showed that 2 out of 9 patients and 8 out of 14 patients were culture positive for Candida spp. (Table 1).\n\nAmong the culture positive patients (n=69 study group; n= 74 control group), the prevalence of oral candidiasis were 33.3% and 10.8% ,while the prevalence of oral colonization were 66.7% and 89.2% among the study and control patients, respectively (Table 2).\n\nAmong the 69 positive cases in the study group, 13 patients exhibited mixed growth of oral yeast, while among the control group 25 patients exhibited mixed growth out of 74. As a result, the numbers of total isolates collected from different specimens from the study group were 92 and 99 isolates from the control group.\n\nPCR products of ITS polymerized region revealed different band size for different species. C. albicans exhibited 500 bp (Figure 1). In contrast, non-C. albicans Candida and less common yeast represented different band sizes ranging from 400–750 bp (Figure 2).\n\nFrom left to right lanes: L, 100bp DNA ladder, L1 negative control, L2,4,5,6 C. albicans (500bp).\n\nFrom right to left lanes: L, 100bp DNA ladder, L1,2,3,6 C. glabrata (750bp), L9,11 C. galbrata (600bp), L4,8 P. kudriavzevii (450bp), L10,12 P. kudriavzevii (400bp), L5,7 C. tropicalis (450bp), L13 L. fermentati (500bp).\n\nDNA sequencing was performed for isolated yeast species (only 90 isolates from 67 out of 69 positive patients in study group and 87 isolates from 66 out of 74 positive patients in the control group). The result of the BLAST sequence analysis for species identification is shown in Table 3.\n\nThe distribution of oral mixed species among the 67 study and 66 control groups is shown in Table 4.\n\nTable 4 shows the distribution of oral mixed species among the (67) study and (66) control groups. It was found that although some patients represented different colonial morphology of mixed oral isolated yeast, they exhibited the same species on sequence identification.\n\nThe identified species were classified according to presence or absence of symptoms of oral candidiasis among the study and control group to detect their association with oral infection and colonization as in Figure 3. This showed that C. albicans was the most common organism associated with oral infection (14 out of 21) and colonization (28 out of 46) among the study group. In the control group it had the same percentage (2/8; 25%) as C. tropicalis and mixed C. glabrata among oral symptomatic patients, whereas it was found to be the most common cause of colonization (21/58; 36.2%).\n\nFrequency of yeast species associated with oral Candida infection (symptoms) and colonization (no symptoms) among the (A) study group and (B) control group.\n\nIdentification of sputum isolates showed that C. albicans was isolated from both oral and sputum specimens from four patients, while three patients exhibited C. albicans from sputum specimens only (oral swabs were negative), and C. tropicalis was isolated from sputum specimen only from one patient. Regarding urine specimens, C. albicans was isolated from both oral and urine samples from one patient in contrast to mixed infections (Pichia kudriavzevii and Pichia kudriavzevii) that were isolated from oral swabs. C. glabrata was also isolated from the urine of another patient.\n\n\nDiscussion\n\nIn the present study it was found that oral Candida isolates were obtained from 69 (41.1%) patients who were under treatment with chemo and/or radiotherapy treatments and from 74 (44.8%) patients without cancer treatment. Xu et al. (2013) found that oral infection was prevalent in 46% (391/850) of all cancer patients, while another study reported the incidence of oral candidiasis ranging from 7 to 52% in cancer patients on chemotherapy and/or radiotherapy (Lone et al., 2014)\n\nThe present study revealed that 8 out of 14 patients were positive for sputum culture among the study group (Table 1). Mohammed et al. (2016) and Ungureanu et al. (2016) reported slightly lower percentages of 30.50% and 33.75%, respectively, in comparison to this study (57.15%). This may be due to differences in study population, since cytotoxic and immunosuppressive therapies promote dissemination of Candida spp. through induction of cytopenias and/or immune cell dysfunction (Blot et al., 2008; Cruciani & Serpelloni, 2008). Also this was expressed in the isolation of 22.2% of Candida species from urine culture of the treated patients among this study, which is relatively similar to Nigar et al. (2016), who found out of 64 culture positive clinical specimens Candida species were identified from 18.75% urine specimens.\n\nAmong the study and control groups, the present study found that oral colonization was significantly (p<0.05) more common than oral Candida infection (66.7% vs 33.3% and 89.2% vs 10.8%, in both groups, respectively). Similar results have been reported by Lone et al. (2014), who found the total colonization to be prevalent in 50% and oral candidiasis in 30% of all cancer patients. In contrast, oral Candida infection was more common (p<0.05) among the study group compared with the control group (33.3% vs 10.8%). A systematic review carried out by Lalla et al. (2010) reported that for all cancer treatments, the weighed prevalence of clinical oral fungal infection was found to be 7.5% pretreatment and 39.1% during treatment. This may be due to chemotherapeutic agents and therapeutic radiation that disrupts the mucosal banner of the mouth, leading to severe oral mucositis, gingivitis, and oral candidiasis. Blasting results of ITS sequence analysis in the present study revealed C. albicans was the most prevalent organism in the two groups (65.6% and 39.1%, study and control groups, respectively). These results are in agreement with previous reports (Ismet et al., 2016 and Aldossary et al., 2016) that studied the prevalence of oral Candida spp and demonstrated that C. albicans was the most prevalent organism.\n\nThe majority of oral infections are due to C. albicans but non-albicans strains, such as C. glabrata and C. tropicalis, have increasingly been implicated in causing disease (Bagg et al., 2003). Similar findings were observed in this study C. tropicalis was found to be the second most common isolate among the study group (14.4%) followed equally (7.8%) by C.glabrata and P. kudriavzevii, whereas among the control group C.glabrata was the second most common isolate (19.5%) followed by C. tropicalis (18.4%).\n\nIt was found that P. kudriavzevii (anamorph of Candida krusei) represented 12.6% among the control group, and P. sporocuriosa was isolated once from the study group only. In addition, C. lusitaniae (teleomorph of Candida lusitaniae) represented 4.6%, and C. dubliniensis represented 1.1% among the control group only (Table 3). These findings are in agreement with Pfaller et al. (2010), who performed a recent ten-year analysis of the worldwide distribution of non-albicans Candida species, and indicated that C. glabrata remains the most common non-albicans species and that C. parapsilosis, C. tropicalis, and C. krusei are also frequently isolated.\n\nLess common yeast species were also detected in small numbers. L. fermentati appeared only once and M. capitatus twice among the study group, while among the control group the later along with S. cervisiae were detected among two patients (2.2%). These findings were in agreement with a study done by Han et al., 2004 and Walsh et al., 2004, who reported that, recently, infections caused by less common yeast species such as Pichia, Rhodotorula, Trichosporon, and Saccharomyces spp. and other rarely encountered species have been reported.\n\nTable 4 shows the presence of identified mixed oral yeast species among the study and control groups. Similarly de Sousa et al. (2016) found the presence of more than one yeast among orogastric cancer patients.\n\nRegarding distribution of species according to presence or absence of symptoms of oral candidiasis, it was found that C. albicans was the most common isolate associated with infection 14/21 (66.7%) and colonization 28/46 (60.9%) among the study group. While among non-albicans Candida, the prevalence of C. tropicalis was 9.5% vs. 2.2% and C. glabrata was 4.8% vs. 4.3% among the oral symptomatic and non-symptomatic study patients, respectively (Figure 3A). Similarly Lone et al., (2014) observed C. albicans to be the most common species (74.39% vs. 65.4%) causing colonization and candidiasis in cancer patients, respectively. Whereas they found C. glabrata was the second most common species followed by C. tropicalis and C. parapsilosis to cause colonization as well as candidiasis in cancer patients. This may be due to geographical region variation.\n\nIn contrast, among the control group C. albicans was detected as the same percentage (2/8; 25%) as C. tropicalis and the mixed C. glabrata among oral symptomatic patients, whereas it represents the most common cause of colonization (21/58; 36.2%) followed by C. glabrata (Figure 3B). This may be due to small number of symptomatic control patients due to the absence of any cancer treatment since it is a predisposing factor for initiation of oral infection by the colonized organisms, mainly by C. albicans, which is the most virulent organism.\n\nAmong the isolated Candida species from sputum and urine specimens, C. albicans was isolated from both oral and sputum specimens from four patients and from both oral and urine from one patient. These patients may have gained the infection or colonization in these organs from their own oral colonized Candida species. Other studies reported that colonized Candida can invade the underlying mucosa and enter the blood stream leading onto disseminated disease with considerable morbidity and mortality if not treated promptly (Lalla et al., 2010 and Shokohi et al., 2011).\n\n\nConclusion\n\nThe present study demonstrates that cancer patients were highly colonized with oral yeast species. C. albicans was the most common isolate associated with oral infection and colonization among the treated cancer patients. In contrast with control group it occupied a higher percent among the colonized species only. As the control group were not under cancer treatment, this lead to oral infection and disseminations to other organs. So early detection and identification of colonized yeast is of great value especially among patients undergoing cancer treatments.\n\nAlthough C. albicans was the most prevalent species, other non-albicans Candida and rarely encountered yeast were also isolated. This indicates that use of proper and accurate molecular methods for yeast identification, especially for unusual yeast species, and prior antifungal treatment as required in cancer patients.\n\n\nData availability\n\nDataset 1: Demographical data of cancer patients. Sheet 1: For case group (168 cancer patients under treatments); Sheet 2: For control group (165 cancer patients); Sheet 3: Pictures demonstrated the colonial morphology for different isolated yeast species. DOI, 10.5256/f1000research.14019.d199685 (Nagla et al., 2018).\n\nSequences from the patients are available on GenBank under accession numbers: MH037201 to MH037237, MH019244 to MH019255, MH016295-MH016371, MH061321-MH061334, MH084778-MH084790, MH016252 to MH016274 and MH104613.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe project was financially supported by Ministry of Higher Education and Scientific Research, Sudan.\n\n\nSupplementary material\n\nSupplementary File 1: Demographic questionnaire.\n\nClick here to access the data.\n\n\nReferences\n\nAkapan A, Morgan R: Oral candidiasis. Postgard Med J. 2002; 78(922): 455–459. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Dwairi ZN, Darwazeh MG, Shukri LA: Isolation of Candida species from the oral cavity and fingertips of complete and partial dentures wearers. J Dent Health Oral Disord Ther. 2014; 1(3): 1–6. Publisher Full Text\n\nAldossary MA, Almansour NA, Abdulraheem BS: Isolation and identification of Candida species from the oral cavity of cancer patients undergoing chemotherapy in Basrah, Irag. J Of Biol Agricul And Healthca. 2016; 6(18). Reference Source\n\nBagg J, Sweeney MP, Lewis MA, et al.: High prevalence of non-albicans yeasts and detection of anti-fungal resistance in the oral flora of patients with advanced cancer. Palliat Med. 2003; 17(6): 477–81. PubMed Abstract \| Publisher Full Text\n\nBlot S, Dimopoulos G, Rello J, et al.: Is Candida really a threat in the ICU? Curr Opin Crit Care. 2008; 14(5): 600–604. 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F1000Research. 2018. Data Source\n\nNigar I, Tarafder S, Khan RR, et al.: Species identification of Candida isolated from clinical specimens in a tertiary care hospital. BSMMU J. 2016; 9(1): 20–25. Publisher Full Text\n\nPfaller MA, Diekema DJ, Gibbs DL, et al.: Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-year analysis of susceptibilities of Candida Species to fluconazole and voriconazole as determined by CLSI standardized disk diffusion. J Clin Microbiol. 2010; 48(4): 1366–1377. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPfaller MA, Diekema DJ, Messer SA, et al.: In vitro activities of caspofungin compared with those of fluconazole and itraconazole against 3,959 clinical isolates of Candida spp., including 157 fluconazole-resistant isolates. Antimicrob Agents Chemother. 2003; 47(3): 1068–1071. 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Reference Source\n\nUngureanu A, Gaman AE, Turculeanu A, et al.: Incidence and Antifungal Susceptibility of Candida albicans infections. Curr Health Sci J. 2016; 42(2): 164–168. Publisher Full Text\n\nWalsh TJ, Groll JA, Hiemenz JE, et al.: Infections due to emerging and uncommon medically important fungal pathogens. Clin Microbiol Infect. 2004; 10 Suppl 1: 48–66. PubMed Abstract \| Publisher Full Text\n\nWhite TJ, Bruns T, Lee S, et al.: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In MA Innis, DH Gelfand, JJ Sninsky and TJ White (ed.), PCR protocols: a guide to methods and applications. Academic Press, Inc., San Diego, Calif. 1990. Reference Source\n\nWilson LS, Reyes CM, Stolpman M, et al.: The direct cost and incidence of systemic fungal infections. Value Health. 2002; 5(1): 26–34. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "40051", "date": "02 Nov 2018", "name": "Dimitrios Farmakiotis", "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 study, but it is rather poorly analyzed. Also, some of the definitions need to be readdressed.\n\nFirst of all, even in cancer patients, growth of Candida from sputum or urine does not mean dissemination; rather colonization. Even if the patients have symptoms, Candida UTIs and even less pneumonia are extremely rare. The authors need to provide solid data such as histopathology, define that there were no other potential pathogens, and symptoms resolved only after antifungal.\n\nI am not sure how appropriate it is to use chi-square to document that one condition (colonization) is more frequent than another (infection) within the same group. Besides, so what? If infection is properly defined, the authors should investigate if there was a statistically significant difference in % between their \"case\" and \"control\" groups.\n\nSpeaking of infection, any cases of candidemia? This is the main clinical syndrome that can result from heavy Candida colonization, mucosal breech, foreign bodies, especially lines, immunosuppression, elimination of other flora with antibacterials etc.\n\nMore clinical information is required and needs to be properly analyzed (e.g. risk factors for non-albicans species, different clinical syndromes etc.).\n\nIt might be good to analyze the data on different species presented in the article tables for statistically significant differences in the frequencies of different Candida species.\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?\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? No", "responses": [ { "c_id": "4105", "date": "07 Nov 2018", "name": "nagla masaad", "role": "Author Response", "response": "The first point of referee comment. Answer: I didn't confirm that it was dissemination. I suggested it may be dissemination due to the presence of the same strain in the oral cavity and urine or sputum in symptomatic patients so I recommend for further investigation so as to confirm it. Point number 2: The authors stated that there was a statistical significant difference between infection and colonization among the case and control group. Point number 4: It would be good to investigate that but, it wasn't one of my objectives.Thanks for your good comment. Nagla" } ] }, { "id": "41097", "date": "30 Jan 2019", "name": "Lucia Bulacio", "expertise": [ "Reviewer Expertise Mycology" ], "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 subject is very interesting since the early study of the epidemiology of infectious diseases in immunocompromised patients contributes to reduced frequency of complications due to invasion and dissemination, which cause increased morbidity and mortality of patients.\nSaccharo genus does not exist (Saccaromyces?) C. dobliensis does not exist (C. dubliniensis?) Pichi kudriavzevii does not exist (Picchia?) P. sporocuriosa? L. fermentati?\nThe first time they are mentioned in the text, all isolates must be named using the complete nomenclature; this is the name of the genus followed by the specific epithet, e.g. Candida albicans, Saccharomyces cerevisiae.\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/7-443
https://f1000research.com/articles/7-441/v1	10 Apr 18	{ "type": "Case Report", "title": "Case Report: Successful treatment of refractory high-flow priapism in a patient with sickle cell disease by selective trans-catheter embolization using an autologous blood clot: A case report", "authors": [ "Muhammad Tahir", "Hiba A. Abbas", "Tariq Tassadaq", "Hiba A. Abbas", "Tariq Tassadaq" ], "abstract": "Priapism is an abnormal prolonged and persistent penile erection lasting more than 4 h, unrelated to sexual desire, stimulation or activity. The three types of priapism are low-flow, high-flow and stuttering. Patients with sickle cell disease (SCD) have increased risk of low-flow and stuttering priapism, but high-flow priapism is relatively uncommon in SCD. We report a case of non-traumatic refractory high-flow priapism evolving from a stuttering low-flow priapism in a patient with SCD. The patient was successfully treated by super-selective transcatheter embolization of the penile arteries with an autologous blood clot. It is proposed that the super-selective transcatheter embolization of unilateral or bilateral penile arteries with autologous blood clot is a relatively safe and effective non-surgical treatment option for high-flow priapism, even in patients with SCD, and has a low probability of developing erectile dysfunction.", "keywords": [ "sickle cell", "priapism", "autologous", "embolization." ], "content": "Introduction\n\nPriapism is an abnormal prolonged and persistent penile erection lasting for more than 4 h that is unrelated to sexual desire, stimulation or activity1–5. Priapism is categorized into three types: low-flow (ischemic, veno-occlusive), high-flow (non-ischemic, arterial), and stuttering (recurrent or intermittent ischemic)5. The low-flow or ischemic form is painful and is the commonest type of priapism (95%)4. High-flow or non-ischemic priapism is rare, painless and is commonly associated with pelvic, perineal or direct penile trauma due to injury to the cavernous artery6–9. Stuttering priapism is ischemic in nature associated with multiple recurrent intermittent self-limiting episodes of persistent erection usually lasting less than 3–4 h and its commonest cause is sickle cell disease (SCD)5. Patients with SCD have an increased risk of low-flow and stuttering priapism, but high-flow priapism is relatively uncommon in these patients5.\n\nWe report a case of non-traumatic refractory high-flow priapism evolving from a stuttering low-flow priapism in a SCD patient. The patient was successfully treated by super-selective transcatheter embolization of the penile arteries with an autologous blood clot.\n\n\nCase report\n\nA 37-year-old patient, who was known to have SCD, glucose-6-phosphate dehydrogenase deficiency and hypertension, presented with priapism. Initially, he developed self-limiting intermittent episodes of sustained erection without sexual excitation for 3 months. Each episode lasted for less than 2 h. Eventually he presented with a sustained erection for 12 days duration in another facility, where he received treatment for low-flow priapism by repeated corporal aspirations and transfusion of 3 units of blood with no detumescence. He was then referred to our hospital with refractory priapism associated with SCD.\n\nThe patient’s blood results revealed a hemoglobin level of 8.9 g/dl (normal, 14–18 g/dl), with 94.7% hemoglobin S. The patient was treated with intravenous hydration and alkalization, nasal oxygen and exchange transfusion. Aspiration of the corpora revealed bright red blood. The patient did not have any significant penile pain at any stage. Color Doppler ultrasound imaging demonstrated a marked increase in the flow of the penile arteries. There were no features of arterio-cavernous fistula or psuedoaneurysm.\n\nAfter discussing the possibility of impotence, the patient agreed for selective embolotherapy. Following the obtainment of written informed consent, pelvic digital subtraction angiography was performed via a right transfemoral artery approach. A 5 French vascular access sheath was placed in the right common femoral artery and a 5 French C2 catheter was engaged in the right internal iliac artery. A 2.4 French microcatheter was advanced coaxially into the ipsilateral internal pudendal artery, which was embolized with an autologous blood clot (Figure 1 and Figure 2). The C2 catheter was then engaged in to the contralateral left internal iliac artery using the cross-over technique and the left internal pudendal artery was embolized with an autologous blood clot after selective catheterization with a microcatheter (Figure 3 and Figure 4).\n\nThere was incomplete detumescence of the penis. Mild tumescence was expected due to considerable cavernous tissue fibrosis. Reduced blood flow was seen in the penile arteries on color Doppler ultrasound after transcatheter embolization. There were no further episodes of priapism and he had adequate self-limiting erections for intercourse.\n\n\nDiscussion\n\nIschemic priapism is an emergency due to the potential risk of developing permanent erectile dysfunction, whereas non-ischemic priapism can be treated conservatively or less aggressively10. Patient history, physical examination, aspirated blood gases and penile Doppler ultrasonography help to categorize priapism into ischemic and non-ischemic types for its appropriate management. In the ischemic form of the priapism, fully rigid corpora cavernosa; relative sparing or little involvement of the corpus spongiosum and glans penis; hypoxic and dark aspirated corporal blood; and absent or minimal arterial blood flow are seen10. In the non-ischemic type, the corpora cavernosa are not fully rigid, the aspirated corporal blood is bright red without hypoxia or acidosis and is associated with increased blood flow, arteriolar–sinusoidal fistula or pseudoaneurysm10. Doppler ultrasound study of penile arteries can be helpful in cases with equivocal clinical findings, where mean and peak systolic velocities can differentiate between ischemic and non-ischemic forms11.\n\nLow-flow priapism is initially managed with intravenous hydration, alkalization, analgesia and exchange transfusion4. However, if priapism persists, further treatment includes corporeal blood aspiration, irrigation with non-heparinized saline and intracorporeal administration of alpha-adrenergic agonists (sympathomimetic agents such as phenylephrine)4. The surgical shunt procedure is the last resort when all attempts of other non-surgical treatment options have failed4,12. The initial management of high-flow priapism is observation and two-thirds of high-flow priapism patients resolve spontaneously during observation13,14. In the rest of the patients with high-flow priapism, embolotherapy is a frequent treatment option, which can be performed in a number of way, including by use of autologous blood clot, gel foam, platinum microcoils or acrylic glue10,15–17. Complications of embolization can include permanent erectile dysfunction, penile gangrene, inadvertent migration of the embolization material into other regional arteries, gluteal ischemia and perineal abscesses18. Autologous blood clot embolotherapy has been reported in post-traumatic high-flow priapism patients19. In our case, after the failure of initial management by corporeal aspiration, the non-traumatic high-flow state was successfully treated using autologous blood clot embolotherapy. The autologous blood clot and gelatin foam have transient effect as embolization agents20 and their use in high-flow priapism patients has the theoretical advantage of recanalization of penile arteries, as compared to platinum microcoils, to reduce the risk of permanent erectile dysfunction. Embolization can be repeated in cases of recurrence. If embolotherapy fails, surgical management is the last treatment option21.\n\n\nConclusions\n\nSuper selective embolization of unilateral or bilateral penile arteries with autologous blood clot is a relatively safe and effective non-surgical treatment option for high-flow priapism, even in patients with SCD and has a low risk of erectile dysfunction.\n\n\nConsent\n\nThe patient provided written informed consent for the publication of this case report.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "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\nMorrison BF, Burnett AL: Stuttering Priapism: Insights into pathogenesis and management. Curr Urol Rep. 2012; 13(4): 268–276. PubMed Abstract \| Publisher Full Text\n\nBurnett AL, Bivalacqua TJ: Priapism: current principles and practice. Urol Clin North Am. 2007; 34(4): 631–642, viii. PubMed Abstract \| Publisher Full Text\n\nBivalacqua TJ, Burnett AL: Priapism: New Concepts in the Pathophysiology and New Treatment Strategies. Curr Urol Rep. 2006; 7(6): 497–502. PubMed Abstract \| Publisher Full Text\n\nBroderick GA, Kadioglu A, Bivalacqua TJ, et al.: Priapism: pathogenesis, epidemiology, and management. J Sex Med. 2010; 7(7): 476–500. PubMed Abstract \| Publisher Full Text\n\nLevey HR, Kutlu O, Bivalacqua TJ: Medical management of ischemic stuttering priapism: a contemporary review of the literature. Asian J Androl. 2012; 14(1): 156–163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBroderick GA: Priapism and sickle-cell anemia: Diagnosis and nonsurgical therapy. J Sex Med. 2012; 9(1): 88–103. PubMed Abstract \| Publisher Full Text\n\nAbujudeh H, Mirsky D: Traumatic high-flow priapism: treatment with super-selective micro-coil embolization. Emerg Radiol. 2005; 11(6): 372–374. PubMed Abstract \| Publisher Full Text\n\nBaba Y, Hayashi S, Ueno K, et al.: Superselective arterial embolization for patients with high-flow priapism: results of follow-up for five or more years. Acta Radiol. 2007; 48(3): 351–354. PubMed Abstract \| Publisher Full Text\n\nHellstrom WJ, Derosa A, Lang E: The use of transcatheter superselective embolization to treat high flow priapism (arteriocavernosal fistula) caused by straddle injury. J Urol. 2007; 178(3 Pt 1): 1059. PubMed Abstract \| Publisher Full Text\n\nShigehara K, Namiki M: Clinical Management of Priapism: A Review. World J Mens Health. 2016; 34(1): 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Stempel C, Zacharakis E, Allen C, et al.: Mean velocity and peak systolic velocity can help determine ischaemic and non-ischaemic priapism. Clin Radiol. 2017; 72(7): 611.e9–611.e16. PubMed Abstract \| Publisher Full Text\n\nWen CC. Munarriz R, McAuley I, et al.: Management of ischemic priapism with high-dose intracavernosal phenylephrine: from bench to bedside. J Sex Med. 2006; 3(5): 918–922. PubMed Abstract \| Publisher Full Text\n\nMontague DK, Jarrow J, Broderick GA, et al.: American Urological Association guideline on the management of priapism. J Urol. 2003; 170(4 Pt 1): 1318–1324. PubMed Abstract \| Publisher Full Text\n\nShrewsberry A, Weiss A, Ritenour CW: Recent Advances in the Medical and Surgical Treatment of Priapism. Curr Urol Rep. 2010; 11(6): 405–413. PubMed Abstract \| Publisher Full Text\n\nHetzichristou D, Salpiggidis G, Hatzimouratidis K, et al.: Management strategy for arterial priapism: therapeutic dilemmas. J Urol. 2002; 168(5): 2074–2077. PubMed Abstract\n\nTakao T, Osuga K, Tsujimura A, et al.: Successful superselective arterial embolization for post-traumatic high-flow priapism. Int J Urol. 2007; 14(3): 254–256. PubMed Abstract \| Publisher Full Text\n\nHakim LS, Kulaksizoglu H, Mulligan R, et al.: Evolving concepts in the diagnosis and treatment of arterial high flow priapism. J Urol. 1996; 155(2): 541–8. PubMed Abstract \| Publisher Full Text\n\nSandock DS, Seftel AD, Herbener TE, et al.: Perineal abscess after embolization for high-flow priapism. Urology. 1996; 48(2): 308–311. PubMed Abstract \| Publisher Full Text\n\nCrummy AB, Ishizuka J, Madsen PO: Posttraumatic priapism: successful treatment with autologous clot embolization. AJR Am J Roentgenol. 1979; 133(2): 329–330. PubMed Abstract \| Publisher Full Text\n\nBurt FB, Schirmer HK, Scott WW: A new concept in the management of priapism. J Urol. 1960; 83: 60–61. PubMed Abstract \| Publisher Full Text\n\nSalonia A, Eardley I, Giuliano F, et al.: European Association of Urology Guidelines on Priapism. Eur Urol. 2014; 65(2): 480–489. PubMed Abstract \| Publisher Full Text" }	[ { "id": "33413", "date": "04 May 2018", "name": "Arthur Burnett", "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\nYou provide an intriguing report and suggest a new treatment consideration for priapism. This specifically applies to the patient who has non-traumatic refractory high-flow priapism that apparently has evolved from a low-flow state in a sickle cell disease patient. Your success is applauded. There are some questions.\n\nSome important data would be helpful to include to convince the reader about your findings. For instance, do you have the data for arterial blood gas measurement from the penis with blood aspiration that correlates with your observation of bright red blood? What were the color Doppler ultrasound imaging parameters that are increased? What were the reduced blood flow parameters after treatment? Perhaps a simple table of before and after blood flow parameters by the ultrasound technique would be very helpful for the reader. A strong suggestion is to include arrows on your figures to show exactly where the embolization was performed. Legends for each figure can then specify the observation based on the location of the arrow. Although this has been a successful technique, it is clear that it has a role specifically in this sort of patient only after high-flow priapism has been confirmed. This would not be a technique that would mistakenly be done for low-flow priapism. Otherwise, serious complications could be expected in the latter condition. It is suggested to the authors to emphasize this point strongly in your conclusion statement.\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? No\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": "33414", "date": "08 May 2018", "name": "David J. Ralph", "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\nI have reviewed the article and here are my comments:\n\nThis patient had the wrong diagnosis made and potentially could have had a disastrous complication of penile gangrene.\nThe diagnosis was ischaemic priapism of 12 days and the initial treatment was correct but deemed a failure.\nAfter aspiration in this context a hyperdynamic flow occurs due to a reactive hyperaemia with reperfusion.\nThe Doppler performed after aspiration therefore records a high flow whereas the perfusion within the small vessels and tissues is absent and is necrotic and later fibroses down.\nTo then embolise a penis that already has necrotic tissue is dangerous.\nThis patient did not have high flow priapism.\n\nIs the background of the case’s history and progression described in sufficient detail? No\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? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/7-441
https://f1000research.com/articles/7-439/v1	10 Apr 18	{ "type": "Software Tool Article", "title": "TCGAbiolinksGUI: A graphical user interface to analyze cancer molecular and clinical data", "authors": [ "Tiago Chedraoui Silva", "Antonio Colaprico", "Catharina Olsen", "Tathiane M Malta", "Gianluca Bontempi", "Michele Ceccarelli", "Benjamin P Berman", "Houtan Noushmehr", "Antonio Colaprico", "Catharina Olsen", "Tathiane M Malta", "Gianluca Bontempi", "Michele Ceccarelli", "Benjamin P Berman" ], "abstract": "The GDC (Genomic Data Commons) data portal provides users with data from cancer genomics studies. Recently, we developed the R/Bioconductor TCGAbiolinks package, which allows users to search, download and prepare cancer genomics data for integrative data analysis. The use of this package requires users to have advanced knowledge of R thus limiting the number of users. To overcome this obstacle and improve the accessibility of the package by a wider range of users, we developed a graphical user interface (GUI) using Shiny available through the package TCGAbiolinksGUI. The TCGAbiolinksGUI package is freely available within the Bioconductor project at http://bioconductor.org/packages/TCGAbiolinksGUI/. Links to the GitHub repository, a demo version of the tool, a docker image and PDF/video tutorials are available from the TCGAbiolinksGUI site.", "keywords": [ "TCGA", "cancer", "genomics", "epigenomics", "bioinformatics" ], "content": "Introduction\n\nThe National Cancer Institute’s (NCI) Genomic Data Commons (GDC), a data sharing platform that promotes precision medicine in oncology, provides a rich resource of molecular and clinical data. As of 2018, almost 13,000 tumor patient samples across 38 different cancer types and subtypes are freely available for download and analysis. Currently, the platform includes data from The Cancer Genome Atlas (TCGA) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET), with the expectation that many other cancer genomic repositories to be incorporated into GDC over the next few years. The publicly available data have been utilized by researchers for novel discoveries and/or validate important findings related to tumorigenesis, improvements in treatment diagnosis and refinement of tumor classifications. To enhance these findings, several important bioinformatics tools to harness genomics cancer data were developed, many of them belonging to the Bioconductor project1.\n\nTCGAbiolinks2, an R/Bioconductor package, was developed to facilitate the analysis of cancer genomics data by incorporating the query, download and processing steps directly from GDC. This tool allows users to advance their data analysis of cancer genomics by harnessing additional Bioconductor packages thereby allowing users access to a wealth of statistical methodologies. In addition, it can perform integrative data analysis across different types of experimental data types, such as DNA methylation and Gene expression data. A detailed comparison between TCGAbiolinks and other bioinformatics tools to analyze cancer genomics data was previously described2. Although TCGAbiolinks is a suitable R package for most data analysts with a strong knowledge and familiarity with R, specifically those who can comfortably write strings of common R commands, we developed TCGAbiolinksGUI to enable user access to the methodologies offered in TCGAbiolinks and to give users the flexibility of point-and-click style analysis without the need to enter specific arguments. TCGAbiolinksGUI takes in all the important features of TCGAbiolinks and offers a graphics user interface (GUI) thereby eliminating any need to be familiar with TCGAbiolinks’ key functions and arguments. In addition, we added new functions to import users’ own raw data for further integrative analysis with GDC data. Tutorials via online documents and YouTube video instructions are available from the website to assist end-users in taking full advantage of TCGAbiolinks.\n\nHere we present TCGAbiolinksGUI, an R/Bioconductor package which uses the R web application framework Shiny3 to provide a GUI to process, query, download, and perform integrative analyses of GDC data.\n\n\nImplementation\n\nThe TCGAbiolinksGUI was created using Shiny, a Web Application Framework for R. TCGAbiolinksGUI incorporates several packages, that provide advanced features to enhance Shiny apps, such as shinyjs to add JavaScript actions4, shinydashboard to add dashboards5 and shinyFiles6 to provide access to the server file system. The following R/Bioconductor packages are used as back-ends for the data retrieval and analysis:\n\n• TCGAbiolinks2 which allows to search, download and prepare data from the NCI’s Genomic Data Commons (GDC) data portal into an R object and perform several downstream analysis;\n\n• ELMER (Enhancer Linking by Methylation/Expression Relationship)7,8 which identifies DNA methylation changes in distal regulatory regions and correlate these signatures with the expression of nearby genes to identify transcriptional targets associated with cancer;\n\n• ComplexHeatmap9 to visualize data as oncoprint and heatmaps;\n\n• pathview10 which offers pathway based data integration and visualization;\n\n• maftools11 to analyze, visualize and summarize genomics MAF (Mutation Annotation Format) files.\n\nThe user interface has been divided into three main GUI menus. The first menu defines the acquisition of GDC data. The second, the ’Analysis’ menu, is subdivided according to the molecular data types. And the third is dedicated to harnessing integrative analyses. Each menu is described below (see Figure 1):\n\n• Data: Provides a guided approach to search for published molecular subtype information, clinical and molecular data available in GDC. In addition, it downloads and processes the molecular data into an R object that can be used for further analysis. For raw DNA methylation data obtained in the form of Intensity Data (IDAT) files, we provide a pipeline using the R/Bioconductor minfi package to prepare the data for subsequent bioinformatics analysis12 performing a background and dye-bias correction with the preprocessnoob function followed by a detection P-value quality masking (sample-specific)13 and probes overlapping repeats or single nucleotide polymorphisms masking (non-sample specific)14 (Figure 3).\n\n• Clinical analysis: Performs survival analysis to quantify and test survival differences between two or more groups of patients and draws survival curves with the ’number at risk’ table, the cumulative number of events table and the cumulative number of censored subjects table using the R/CRAN package survminer15.\n\n• Epigenetic analysis: Performs a differentially methylated regions (DMR) analysis, visualizes the results through both volcano and heatmap plots, and visualizes the mean DNA methylation level by groups or subtypes. For certain tumor types like Glioma, we have added a function to classify non TCGA derived DNA methylation data into one of the 7 published epigenomic subtypes16 using a RandomForest (RF) trained model derived from DNA methylation signatures available from the Cancer Genome Atlas (Figure 4). Description of how the RF models were created can be found in TCGAbiolinksGUI.data vignette.\n\n• Transcriptomic analysis: Performs a differential expression analysis (DEA), and visualizes the results as either volcano or heatmap plots. Pathway analysis can be performed on a list of differentially expressed genes10.\n\n• Genomic analysis: Visualize and summarize the mutations from MAF (Mutation Annotation Format) files through summary plots and oncoplots using the R/Bioconductor maftools package9,11 (Figure 2 and Figure 6).\n\nThe panel on the left shows the menus divided into different analyses, the panel on the right shows the controls available for the selected menu. In the center is a volcano plot window from the analysis menu. It is possible to control the colors, to change cut-offs, to export results into a CSV document and to export the plot.\n\n• Integrative analysis: Integrate the DMR and DEA results through a starburst plot. Integrate clinical and mutation data by way of a Kaplan-Meier survival analysis for groups of mutated samples vs non-mutated for a given gene (Figure 5). DNA methylation and gene expression data can be further analyzed using the R/Bioconductor ELMER package to discover functionally relevant genomic regions associated with cancer7,8.\n\nThis maftools plot shows a summary of the MAF file. Highlighting the most mutated genes, SNV class, and variant classification distributions within a tumor type.\n\nTable lists all files which will be processed. Data retrieved from GEO (accession GSE61160).\n\nPredicting glioma epigenomic molecular subtypes based on DNA methylation using data from GEO (accession GSE61160).\n\nPerforming Kaplan-Meier survival analysis for groups of samples with a mutation in gene USH2A vs WT.\n\nEach column represents a sample and each row a different gene. The top barplot has the frequency of mutations for each patient, while the right barplot has the frequency of mutations in each gene. By default, samples ordered by the most mutated genes.\n\nWe provide a guided tutorial for users via an online vignette document which details each step and menu function. Printable PDF and YouTube video instructions (http://bit.ly/TCGAbiolinksGUI_videoTutorials) are provided to help users utilize TCGAbiolinksGUI. A demonstration version of the tool is available at TCGAbiolinksGUI. To help improve and expand our tool over time, users are encouraged to report and file bug reports or feature requests via our GitHub repository.\n\nWe recognize that one of the possible drawbacks of using our tool is the arduous process of installing the R/Bioconductor environment and all of the required dependencies. One solution would be to host the tool on a server, however the high demand for space, computational processing, and access for multiple users would make this approach financially challenging; instead, we encourage users to use it on their own computers. However, to further simplify the usability and accessibility of our tool, we provide a Docker image file that contains the complete R/Bioconductor environment configured to use TCGAbiolinksGUI. This file is compatible with most popular operating systems and it is available online. This image can be easily downloaded and deployed through the Kitematic tool, a simple application for managing Docker containers for Mac, Linux, and Windows. A detailed documentation of how to obtain and use the docker image via the Kitematic application is available at TCGAbiolinksGUI help document. The Docker image will be updated to coincide with any regular updates of TCGAbiolinksGUI. We believe this will provide several types of access for end-users interested in analyzing cancer genomics data stored at GDC.\n\n\nOperation\n\nThe package can run on any platform with a R version ≥ 3.4 or higher and Bioconductor version ≥ 3.6 and higher.\n\n\nResults and discussion\n\nTo provide end-users with insights and application of TCGAbiolinksGUI; 1) we compare TCGAbiolinksGUI with other published bioinformatics tools and 2) we provide a use-case that allows users a step-bystep guide to analyzing their own cancer molecular data alongside TCGA data.\n\nWeb tools used for cancer data analysis might be classified into two broad groups. The first group only provides an interface to existing software analysis tools. The Galaxy project, which is an open, web-based platform for accessible, reproducible, and transparent computational biomedical research, is an example of such a tool that belongs to this group. The other group is composed of exploratory tools mainly focused on the visualization of processed data and pre-computed results. The cBioPortal project17,18, by providing several visualizations for mining the TCGA data, is an example of a tool that falls within this category.\n\nIf one were to classify TCGAbiolinksGUI, it would belong to the first group. Compared to the Galaxy project, TCGAbiolinksGUI offers an open platform which improves the accessibility of R/Bioconductor packages, allowing users an advantage to integrate their features with existing Bioconductor packages. Unlike the Galaxy project, which requires the interface elements to be structured through XML files19, TCGAbiolinksGUI can resolve this simply because it was built within the R/Shiny framework. cBioPortal and TCGAbiolinksGUI provide users with access to raw and processed data, however TCGAbiolinksGUI allows users to perform in-depth integrative analysis, a functionality which cBioPortal currently lacks. For example, if a user is interested in defining differentially expressed genes or DNA methylation events between two populations of tumors (i.e. FOXA1 mutants and FOXA1 wildtypes), by using cBioPortal, a user would have to download the gene expression, DNA methylation and mutation data, define the samples per group, and import the data into their favorite statistical tool to identify their list of differentially expressed or methylated genes. Whereas, with TCGAbiolinksGUI, we developed the platform so that the user can define the sample groups based on their mutation spectrum, perform supervised analysis that can then define differentially expressed or methylated gene list and this can be directly ported into pathway analysis. In addition, if the user is interested in observing survival differences between the groups, this can also be done within TCGAbiolinksGUI and thereby reducing the need to exit a specific data platform or having to transform the downloaded data to fit some other statistical platform to achieve the same goals.\n\n\nUse case\n\nIn order to illustrate an integrative analysis using TCGAbiolinksGUI, we provide a use case available at https://bioinformaticsfmrp.github.io/Bioc2017.TCGAbiolinks.ELMER/index.html. This use case highlights a step by step guide for one to perform an integrative analysis using TCGA-LUSC (Lung Squamous Cell Carcinoma) data retrieved directly from GDC server20.\n\n\nConclusion\n\nTCGAbiolinksGUI was developed to provide a user-friendly interface of our TCGAbiolinks package. TCGAbiolinksGUI is designed specifically for the least experienced R user to import GDC data and perform R/Bioconductor analysis as well as for the most experienced R user, who could execute several of the R/Bioconductor functions without the need to write several lines of R code. For the R/Bioconductor developers, the package has an extensible design feature that allows users 1) to add new features by modifying a few lines of the main code, 2) to add a file with user interface elements on the client side, and 3) add a file with their control on the server side.\n\nAlso, TCGAbiolinksGUI supports the most updated R/Bioconductor data structures (i.e. SummarizedExperiment and MultiAssayExperiment) which allow handling data and metadata into one single object and validates several integrity requirements. Thereby, TCGAbiolinksGUI package allows data handling to be as efficient as possible and thereby limits and avoids user errors in data manipulation such as sample removal that involves also metadata deletion.\n\nFinally, several efforts to understand genomic and epigenomic alterations associated with tumor development has been made over the last few years, which presents several bioinformatics challenges, such as data retrieval and integration with clinical data and other molecular data types. By creating a graphical interface to tools like TCGAbiolinks whose relevance is seen in various articles21–23, this package will allow end-users to facilitate the mining of cancer data deposited in GDC, in hopes to aid in analyzing and discovering new functional genomic elements and potential therapeutic targets for cancer.\n\n\nData and software availability\n\nTCGAbiolinksGUI is a platform independent R package (R ≥ 3.4) available at: https://doi.org/10.18129/B9.bioc.TCGAbiolinksGUI.\n\nSource code TCGAbiolinksGUI is available at: https://github.com/BioinformaticsFMRP/TCGAbiolinksGUI.\n\nLicense: GNU General Public License version 3 (GNU GPL3)\n\nComplementary data required to execute the package is available at: https://github.com/BioinformaticsFMRP/TCGAbiolinksGUI.data or at https://doi.org/doi:10.18129/B9.bioc.TCGAbiolinksGUI.data24.\n\nSoftware documentation is available at: https://bioconductor.org/packages/devel/bioc/vignettes/TCGAbiolinksGUI/inst/doc/index.html\n\nDetailed steps of the use case are available at: https://bioinformaticsfmrp.github.io/Bioc2017.TCGAbiolinks.ELMER/index.html.\n\nTo install the stable version from the Bioconductor repository http://bioconductor.org/packages/TCGAbiolinksGUI/ please use the following code.\n\n\n\nAnd to install the development version of the package via GitHub:\n\n\n\nThis installation process has been tested on a Debian 9.1 machine (the following libraries had to be installed: libpng-dev and libmariadb-client-lgpl-dev (command: sudo apt-get install libpng-dev libmariadbclient-lgpl-dev).\n\nAlso, due to the number of libraries loaded we had to increase the maximum number of DLL R can load, for more information please check the vignette section \"Increasing loaded DLL\".\n\n\n\n", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work has been supported by a grant from Henry Ford Hospital (H.N.) and by the São Paulo Research Foundation (FAPESP) (2016/01389-7 to T.C.S. & H.N. and 2015/07925-5 to H.N.) the BridgeIRIS project, funded by INNOVIRIS, Region de Bruxelles Capitale, Brussels, Belgium, and by GENomic profiling of Gastrointestinal Inflammatory-Sensitive CANcers (GENGISCAN), Belgian FNRS PDR (T100914F to A.C., C.O.& G.B.). T.C.S. and B.P.B. were supported by the NCI Informatics Technology for Cancer Research program, NIH/NCI grant 1U01CA184826 and Genomic Data Analysis Network NIH/NCI grant 1U24CA210969.\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 are grateful to the OMICs lab and the GDC team for suggestions in the design of TCGAbiolinksGUI interface. We are also grateful for Susan MacPhee for critical review of the manuscript and vignettes.\n\n\nReferences\n\nGentleman RC, Carey VJ, Bates DM, et al.: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004; 5(10): R80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nColaprico A, Silva TC, Olsen C, et al.: TCGAbiolinks: an r/bioconductor package for integrative analysis of TCGA data. Nucleic Acids Res. 2016; 44(8): e71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang W, Cheng J, Allaire JJ, et al.: shiny: Web Application Framework for R. R package version 0.14, 2016. Reference Source\n\nAttali D: shinyjs: Easily Improve the User Experience of Your Shiny Apps in Seconds. R package version 0.9.1, 2017. Reference Source\n\nChang W, Ribeiro BB: shinydashboard: Create Dashboards with ’Shiny’. R package version 0.6.1, 2017. Reference Source\n\nLin PT: shinyFiles: A Server-Side File System Viewer for Shiny. R package version 0.6.2. 2016. Reference Source\n\nYao L, Shen H, Laird PW, et al.: Inferring regulatory element landscapes and transcription factor networks from cancer methylomes. Genome Biol. 2015; 16(1): 105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilva TC, Coetzee SG, Yao L, et al.: Enhancer linking by methylation/expression relationships with the r package elmer version 2. bioRxiv. 2017. Reference Source\n\nGu Z, Eils R, Schlesner M: Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics. 2016; 32(18): 2847–9. PubMed Abstract \| Publisher Full Text\n\nWeijun L, Brouwer C: Pathview: an r/bioconductor package for pathway-based data integration and visualization. Bioinformatics. 2013; 29(14): 1830–1831. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMayakonda A, Koeffler PH: Maftools: Efficient analysis, visualization and summarization of maf files from large-scale cohort based cancer studies. bioRxiv. 2016. Publisher Full Text\n\nAryee MJ, Jaffe AE, Corrada-Bravo H, et al.: Minfi: a flexible and comprehensive Bioconductor package for the analysis of infinium DNA methylation microarrays. Bioinformatics. 2014; 30(10): 1363–1369. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorris TJ, Beck S: Analysis pipelines and packages for infinium humanmethylation450 beadchip (450k) data. Methods. 2015; 72: 3-8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou W, Laird PW, Shen H: Comprehensive characterization, annotation and innovative use of infinium dna methylation beadchip probes. Nucleic Acids Res. 2017; 45(4): e22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKassambara A, Kosinski M: survminer: Drawing Survival Curves using ’ggplot2’. R package version 0.4.0, 2017. Reference Source\n\nCeccarelli M, Barthel FP, Malta TM, et al.: Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma. Cell. 2016; 164(3): 550–563. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGao J, Aksoy BA, Dogrusoz U, et al.: Integrative analysis of complex cancer genomics and clinical profiles using the cbioportal. Sci Signal. 2013; 6(269): pl1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCerami E, Gao J, Dogrusoz U, et al.: The cbio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012; 2(5): 401–404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTuraga N, Freeberg MA, Baker D, et al.: A guide and best practices for r/bioconductor tool integration in galaxy [version 1; referees: 1 approved, 1 approved with reservations]. F1000Res. 2016; 5: 2757. Publisher Full Text\n\nGrossman RL, Heath AP, Ferretti V, et al.: Toward a Shared Vision for Cancer Genomic Data. N Engl J Med. 2016; 375(12): 1109–1112. PubMed Abstract \| Publisher Full Text\n\nBroutier L, Mastrogiovanni G, Verstegen MM, et al.: Human primary liver cancer-derived organoid cultures for disease modeling and drug screening. Nat Med. 2017; 23(12): 1424–1435. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhassemi S, Vejdovszky K, Sahin E, et al.: Fgf5 is expressed in melanoma and enhances malignancy in vitro and in vivo. Oncotarget. 2017; 8(50): 87750–87762. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLetellier E, Schmitz M, Ginolhac A, et al.: Loss of myosin vb in colorectal cancer is a strong prognostic factor for disease recurrence. Br J Cancer. 2017; 117(11): 1689–1701. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilva TC, Colaprico A, Olsen C, et al.: TCGAbiolinksGUI: A Graphical User Interface to analyze cancer molecular and clinical data. bioRxiv. Bioinformatics - Submitted for review. 2017. Publisher Full Text" }	[ { "id": "33002", "date": "23 Apr 2018", "name": "Zuguang Gu", "expertise": [ "Reviewer Expertise Bioinformaitcs", "next generation sequencing", "R packages development", "visualization" ], "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\nSilva et al. developed a new package TCGAbiolinksGUI which provides an easy way to query, download and analyze TCGA datasets. It would be a useful tool for bioinformaticians also non-bioinformaticians to process and get use of the massive TCGA datasets.\nBesides taking care of downloading the TCGA datasets, TCGAbiolinksGUI package also provides basic functions/plots to process mutation/expression/methylation datasets. Additionally, TCGAbiolinksGUI performs integrative analysis of methylation and gene expression and does motif finding on the inferred regulatory network.\nThe TCGAbiolinksGUI has a very modern UI design, quite sophisticated programming and very friendly user interface. The structure of the analysis workflow is very clear. Besides that, it also has very detailed documentations even with videos. For the functionalities TCGAbiolinksGUI has already implemented, basically they are nice and I don't have major issues on it.\nFor some reason, I only tested TCGAbiolinksGUI with version 1.4.7 (the release version on Bioc at the time of reviewing this paper) and R version 3.4.4. I had some errors when testing some of the functionalities for which I think it should due to the lower version I was using and I would expect they should work OK with the development version.\nFollowing are my minor comments:\n1. There are some low-level errors which cause TCGAbiolinksGUI() function crashed and the webpage closed. E.g. when I tried to use \"network inference\" or \"maftools plots\", it gave error \"no minet function/no read.maf function\". I would guess it's mainly due to I was using the old version of this package. But it would be nice to capture these low-level errors also and print them in the web interface without stopping `TCGAbiolinksGUI()`. Also in DEA analysis, if group column is not set, `TCGAbiolinksGUI()` stops.\n2. In many analysis where \"group column\" is needed, there are so many \"clinical information fields\" in the drop-down list. Is it possible to remove some of them? e.g. sample Ids or columns with too many missing values, or these numeric columns which I think they would never be used in group comparisons.\n3. When a file is selected, there is no information on the web interface to tell users whether the file is selected or which file is selected.\n4. For DEA analysis, if group levels which are used for comparison are forgot to provides, the error information is not informative (\"Each group should have at least one sample\")\n5. When I do enrichment analysis for differentially expressed genes, I directly used the file generated at the \"DEA analysis\" step. However, it gave the error \"no Gene_symbol column\", but I checked there does have a \"Gene_symbol\" column (which is the first column in the file). I guess it might due to I was using the old version of the package.\n6. When making heatmaps for different genes or DMRs, it is possible to put the grouping information which was used for comparision as default column annotation? I think it won't be too difficult because the group column and group levels are encoded in the file name of DEA or DMR file.\n7. Is it possible to export the R code of making each plot (or performing each analysis)? Users can use these R scripts as template and customize later. E.g. since there is no option to configure the annotation colors, with the R script, users can adjust this part by themselves later.\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? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "33000", "date": "25 Apr 2018", "name": "Michael Lawrence", "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 major concern is that this seems like a GUI that is doing a lot of different things and thus should be more modular. The authors emphasize the connection to GDC, but many of the features are applicable to any similar dataset. Ideally, each panel would be its own Shiny module (or maybe even a separate package entirely), and expert users and app developers could select specific modules and integrate them with custom modules to create new applications. There is mention of extensibility in the text. Does that rely on the Shiny module system? It would be helpful for the reader to know that if true. The authors actually compare their tool to Galaxy, a highly modular system. GUIs have a tendency to be monolithic; we should resist that in Bioconductor. This package has 181 total dependencies!\nAnother point is that GUIs for exploratory data analysis should not only be useful for novices in a programming language. There are times when a GUI is more convenient than programming, even for an expert programmer. A GUI provides an alternative interface to the command line, thus opening the underlying functionality to other use cases, whether a bench biologist desperate for a way to see the data, or a computational biologist who wants to quickly explore the data visually while implementing a more sophisticated analysis.\nWhy not include the use case / workflow in the publication itself? It's useful to have an archive of that.\nSome minor points:\nThe abstract mentions a website but does not link to it (until maybe later) The use of the word \"advanced\" to describe the R users of TCGABiolinks is ambiguous. What exactly do you mean by \"advanced\"? Later on, advanced appears to be mean anyone who can write simple R code. Maybe just drop the ambiguous adjective? \"Gene expression\" do not capitalize \"Gene\" The phrase \"specifically those who can comfortably write strings of common R commands\" is awkward, especially since R does not really have \"commands\". Maybe say something like: \"Although TCGAbiolinks is accessible to data analysts who are familiar with R programming, ...\", although I would phrase it more positively, rather than as deficiency of TCGAbiolinks, which is just playing the role that it is meant to play in a larger framework. \"graphics user interface\" should be \"graphical user interface\" \"Web Application Framework\" - no need to capitalize\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/7-439
https://f1000research.com/articles/6-1722/v1	22 Sep 17	{ "type": "Software Tool Article", "title": "Fragger: a protein fragment picker for structural queries", "authors": [ "Francois Berenger", "David Simoncini", "Arnout Voet", "Rojan Shrestha", "Kam Y.J. Zhang", "Francois Berenger", "David Simoncini", "Arnout Voet", "Rojan Shrestha" ], "abstract": "Protein modeling and design activities often require querying the Protein Data Bank (PDB) with a structural fragment, possibly containing gaps. For some applications, it is preferable to work on a specific subset of the PDB or with unpublished structures. These requirements, along with specific user needs, motivated the creation of a new software to manage and query 3D protein fragments. Fragger is a protein fragment picker that allows protein fragment databases to be created and queried. All fragment lengths are supported and any set of PDB files can be used to create a database. Fragger can efficiently search a fragment database with a query fragment and a distance threshold. Matching fragments are ranked by distance to the query. The query fragment can have structural gaps and the allowed amino acid sequences matching a query can be constrained via a regular expression of one-letter amino acid codes. Fragger also incorporates a tool to compute the backbone RMSD of one versus many fragments in high throughput. Fragger should be useful for protein design, loop grafting and related structural bioinformatics tasks.", "keywords": [ "protein fragments", "protein design", "fragments database", "structural query", "triangular inequality" ], "content": "Introduction\n\nNowadays, a large number of protein structures are available (122,761 as of July 2017 at RCSB) and protein fragments are frequently used in structural bioinformatics. Protein structure prediction methods such as Rosetta1, QUARK2 and EdaFold3,4 use protein fragments as building blocks. Protein fragments are also used in crystallographic phasing5–7 and model rebuilding8. The quality of protein models can be improved by combining protein fragments with molecular dynamics9. Other applications include the curation of unresolved loops in crystal structures10,11, grafting of loop sequences on protein scaffolds and other protein design algorithms12,13.\n\nSome fragment pickers14,15 and protein fragment databases16,17 are currently available. Of particular interest is the Super method15 that uses the lower bound of RMSD18 to screen the whole fragment space. However, our research on protein design and refinement of protein decoys for crystallographic phasing required specific options and therefore a new fragment picker.\n\n\nMethods\n\nInput: d b: fragment set to query\n\nInput: r f : reference fragment set\n\nInput: q: query fragment\n\nInput: dq: RMSD threshold\n\nOutput: mf : matching fragment set\n\nmf ← d b\n\n{fuzzy query: prune the fragment space}\n\nfor rj in r f do\n\nd ← distance(q, rj)\n\ndinf ← d – dq\n\ndsup ← d + dq\n\nmf ← {∀fi ∈ mf \| distance(fi, rj) ∈ [dinf, dsup]}\n\n{distance (fi, rj) comes from the database index}\n\nend for\n\n{exact query: refine the result of pruning}\n\nmf ← {∀fi ∈ mf \| distance(fi, q) ≤ dq}\n\nreturn mf\n\nFragger exploits the triangular inequality of RMSD19 to prune the fragment space (Figure 1 and Algorithm 1). RMSDs are computed efficiently via the QCP method20. Fragger is written in OCaml21, except backbone RMSD computations which are performed with a new version of the C++ ranker tool from Durandal22. Computations are parallelized on multi-core computers via the Parmap library23.\n\nFragger allows a database to be queried with a fragment and an RMSD threshold. Matching fragments are ranked by RMSD to the query. Fragger’s ranker tool allows to compute the backbone RMSD of a single fragment versus many. Fragger can deal with residue gaps or a selection of residues from the query, create a fragment database from a set of Protein Data Bank (PDB) files, work with all fragment lengths and extract specific or randomly-chosen fragments from a database.\n\nq is at distance d1 (resp. d2) from reference fragment r1 (resp. r2). Only fragments which are both within d1 ± dq of r1 and d2 ± dq of r2 will undergo an RMSD calculation. Middle: 13 residues loops that can connect residue ALA 98 to GLY 110 in chain A of PDB 1MEL. The query loop is shown in red. Only its first and last three residues were used to rank the retrieved fragments. Right: Backbone of PDB 1BKR covered with ten residue fragments from non-homologous proteins retrieved with Fragger.\n\nCompared to existing fragment pickers, some of the specific functionalities required by users include:\n\nOutputing only the N best or N first found fragments matching a query (this can make a query terminate faster)\n\nConstraining the amino acid sequences allowed to match a query (for loop grafting)\n\nReading and writing PDB fragments from/to a binary format (faster than reading/writing regular PDB files)\n\nPreventing a list of PDB codes from matching a query\n\nAutomatically varying the RMSD threshold to the query until a given number of fragments is reached.\n\nUsers need to install OPAM and the pdbset command from CCP4 in order to use Fragger.\n\nDetails on how to install Fragger and usage examples are provided in the README file of the released software.\n\n\nResults and discussion\n\nTests were performed on one core of a 2.4GHz Intel Xeon workstation with 12GB of RAM running Ubuntu Linux 12.04. The PDB dataset is composed of all proteins determined by X-ray, without highly similar sequences (30% sequence identity cutoff) in order to create a challenging set of fragments to benchmark a protein design algorithm. It contains 13,554 PDBs. PDBs were extracted from the protein data bank website using the advanced search tab and ticking the \"Retrieve only representatives at 30% sequence identity\" box. Querying with a three (resp. nine) residues fragment takes at least 6.75s (resp. 5.2s). Query time varies with the query fragment, reference fragments and RMSD threshold to the query. Reference fragments can be chosen randomly. Pruning of the search space is better if there are at least three reference fragments and they are far from each other. For one time tasks, it is not necessary to create RMSD indices and actually query a database, as fragments extraction and RMSD computations are fast enough. For example, it takes only 15s to generate all (41,200) fragments of 13 residues starting with alanine and ending with glycine (middle of Figure 1). Ranking them to the query takes 1.5s. When working on PDB files, the ranker tool included with Fragger can compute 66,580 (resp. 23,784) RMSD/s on the backbone of three (resp. nine) residue fragments. These numbers become 304,149 (resp. 138,744) RMSD/s when working on Fragger’s binary-encoded PDBs. In the future, it might be possible to improve the performance of Fragger by incorporating a faster score than RMSD, such as BCscore24.\n\nFragger can be useful for protein design, loop grafting and retrieval of candidates to rebuild low-confidence regions of protein models.\n\n\nSoftware availability\n\nFragger can be downloaded from: https://github.com/UnixJunkie/fragger or http://www.riken.jp/zhangiru/software/fragger.tgz.\n\nArchived source code at the time of publication: https://zenodo.org/record/87732025\n\nSoftware license: LGPL.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the “Initiative Research Unit” program from RIKEN, Japan, the Japanese Society for the Promotion of Science (JSPS) and computing resources on the RIKEN Integrated Cluster of Clusters (RICC). FB is a JSPS international fellow.\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\nLeaver-Fay A, Tyka M, Lewis SM, et al.: Rosetta3: An Object-Oriented Software Suite for the Simulation and Design of Macromolecules. Methods Enzymol. Academic Press, 2011; 487: 545–574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu D, Zhang Y: Ab initio protein structure assembly using continuous structure fragments and optimized knowledge-based force field. Proteins. 2012; 80(7): 1715–1735. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimoncini D, Berenger F, Shrestha R, et al.: A Probabilistic Fragment-Based Protein Structure Prediction Algorithm. PLoS One. 2012; 7(7): e38799. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimoncini D, Schiex T, Zhang KY: Balancing exploration and exploitation in population-based sampling improves fragment-based de novo protein structure prediction. Proteins. 2017; 85(5): 852–858. PubMed Abstract \| Publisher Full Text\n\nRodriguez DD, Grosse C, Himmel S, et al.: Crystallographic ab initio protein structure solution below atomic resolution. Nat Methods. 2009; 6(9): 651–653. PubMed Abstract \| Publisher Full Text\n\nShrestha R, Simoncini D, Zhang KY: Error-estimation-guided rebuilding of de novo models increases the success rate of ab initio phasing. Acta Crystallogr D Biol Crystallogr. 2012; 68(Pt 11): 1522–1534. PubMed Abstract \| Publisher Full Text\n\nShrestha R, Zhang KY: A fragmentation and reassembly method for ab initio phasing. Acta Crystallogr D Biol Crystallogr. 2015; 71(Pt 2): 304–312. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVanhee P, Verschueren E, Baeten L, et al.: BriX: a database of protein building blocks for structural analysis, modeling and design. Nucleic Acids Res. 2011; 39(Database issue): D435–D442. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBudowski-Tal I, Nov Y, Kolodny R: FragBag, an accurate representation of protein structure, retrieves structural neighbors from the entire PDB quickly and accurately. Proc Natl Acad Sci U S A. 2010; 107(8): 3481–3486. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTramontano A, Lesk AM: Common features of the conformations of antigen-binding loops in immunoglobulins and application to modeling loop conformations. Proteins. 1992; 13(3): 231–245. PubMed Abstract \| Publisher Full Text\n\nSteipe B: A revised proof of the metric properties of optimally superimposed vector sets. Acta Crystallogr A. 2002; 58(Pt 5): 506. 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Data Source" }	[ { "id": "26264", "date": "11 Oct 2017", "name": "Pierre Tuffery", "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 describes an approach to quickly scan large collection of proteins to identify fragments similar to a request. Not considering indels, this approach is, as stated by the authors, in the context of fragment grafting, loop modeling, protein design or crystallographic phasing.\nThe metrics used to quantify the similarity is that of the RMSd. The rationale here is to to use the triangular inequality of RMSd to setup a two step procedure:\n- decompose the complete set of fragments present in the collection of proteins by as a limited subset of representative fragments - quickly identify the representative fragments similar to the query in a way to perform effective pruning of the complete collection of fragments, ensuring not discarding the matching fragments, and then perform a systematic search for the fragments of the classes associated with the matching representative fragments.\nThis kind of approach has been used in several contexts and is interesting. The manuscript however could easily be improved.\nHere are some specific comments:\n- The introduction could benefit from a better description of the rationale underlying Fragger, including its use in different contexts. For instance, such a strategy has also been used for the fast similarity search of small compounds.\n- The introduction could benefit from a larger overview of the approaches that have been setup to address questions similar to that of Fragger. There are also a series of web servers focusing on this goal that are not cited.\n- The way the algorithm is described makes it rather uneasy to understand. There could first be some awkwardness in the notations. For instance, in the algorithm description, blanks between d and b and between r and f could be discarded. Secondly, it could be difficult for a reader to understand the role of the representative fragments, the way they are identified and used from the present description of the algorithm. Probably an additional flowchart or figure to explain it would be welcome.\n- The critical parameters of the procedure are not really identified. What are the effective cutoff values, how do they impact on the search ?\n- It seems Fragger offers possibilities to constrain amino acidd sequences. Is it a prior or a posterior filtering ?\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? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "3574", "date": "10 Apr 2018", "name": "Kam Zhang", "role": "Author Response", "response": "> - The introduction could benefit from a better description of the > rationale underlying Fragger, including its use in different contexts. For > instance, such a strategy has also been used for the fast similarity > search of small compounds. We have added an extra paragraph in the introduction to mention related methods found in chemoinformatics. > - The introduction could benefit from a larger overview of the > approaches that have been setup to address questions similar to that of > Fragger. There are also a series of web servers focusing on this goal > that are not cited. We have added several citations to web servers and protein fragment databases which are using various methods. > - The way the algorithm is described makes it rather uneasy to > understand. There could first be some awkwardness in the notations. For > instance, in the algorithm description, blanks between d and b and > between r and f could be discarded. Secondly, it could be difficult > for a reader to understand the role of the representative fragments, > the way they are identified and used from the present description of > the algorithm. Probably an additional flowchart or figure to explain it > would be welcome. We have renamed some variables in the algorithm to bypass typographic problems introduced by the journal's style-sheet. We have also added a new paragraph and one reference about choosing good reference fragments. > - The critical parameters of the procedure are not really identified. What > are the effective cutoff values, how do they impact on the search ? This is quite complex: the search speed is influenced by the protein database, the fragment length and the query RMSD tolerance. In the manuscript, we now summarize the general trend as: \"In general, the longer the required fragment length and the smaller the RMSD tolerance, the faster the query.\". > - It seems Fragger offers possibilities to constrain amino acidd > sequences. Is it a prior or a posterior filtering? We updated the manuscript to indicate that this is done after geometric filtering." } ] }, { "id": "28334", "date": "18 Jan 2018", "name": "Charlotte Deane", "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 paper, the authors describe Fragger, a web server for retrieving fragments from a database of structures based on a query fragment. Their program allows for customisation in terms of number of fragments output and in terms of sequence constraints.\nThe paper is made up of previously published methods put together as a potentially useful package.\n\nThe Super method (mentioned in the introduction of the manuscript) seems to fulfil the same purpose as Fragger. No comparison is provided between the two methods in terms of performance and quality of fragments output.\nIt is unclear from the Methods section how the reference fragment set is selected. It is mentioned in the Results that these fragments can be selected at random, but the authors never discuss whether this choice can have an impact on the performance of the algorithm.\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? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "3575", "date": "10 Apr 2018", "name": "Kam Zhang", "role": "Author Response", "response": "> The Super method (mentioned in the introduction of the manuscript) seems > to fulfil the same purpose as Fragger. No comparison is provided between > the two methods in terms of performance and quality of fragments output. Super uses only CA to calculate RMSD. Fragger uses backbone atoms to calculate RMSD, since our users want to preserve secondary structure information. Since Super uses four times fewer atoms than Fragger for each RMSD calculation,we don't think such a comparison would be fair. > It is unclear from the Methods section how the reference fragment set > is selected. It is mentioned in the Results that these fragments can be > selected at random, but the authors never discuss whether this choice > can have an impact on the performance of the algorithm. Indeed, good reference fragments can improve the search performance. We have added a new paragraph and one reference about choosing good reference fragments." } ] } ]	1	https://f1000research.com/articles/6-1722
https://f1000research.com/articles/6-1964/v1	07 Nov 17	{ "type": "Case Report", "title": "Case Report: An incidental finding of an adrenal metastases noted in a “collision tumor” from a large malignant nerve sheath tumor of the thigh", "authors": [ "Matthew D. Shaines", "Shitij Arora", "Matthew D. Shaines" ], "abstract": "In cases of peripheral nerve sheath tumors, current guidelines do not recommend routine abdominal imaging to stage the disease, as extra-pulmonary metastasis is considered rare. We report a case of large peripheral nerve sheath tumor in a 40 year-old-female with neurofibromatosis type 1 who had isolated adrenal metastasis. She underwent primary and adrenal metastasis resection.", "keywords": [ "nerve sheath tumor", "adrenal", "lung", "metastasis" ], "content": "Introduction\n\nSoft tissue sarcomas of the extremity are a rare, comprising less than 1% of all malignancies1. These sarcomas are a histologically heterogeneous group of tumors with a predilection for hematogenous spread. Distant metastatic disease is found in approximately 20–30% of patients1–3, with pulmonary lesions accounting for 75% of these cases1,4,5. Because of the relatively low incidence of extra-pulmonary metastasis, the current guidelines from the National Comprehensive Cancer Network (NCCN) recommend to consider abdominal/pelvic CT imaging for certain subgroups of sarcomas (myxoid/round cell liposarcoma, epithelial sarcoma, angiosarcoma, leiomyosarcoma)6. A similar recommendation is made by the European Society of Medical Oncology (ESMO)/European Sarcoma Network Working Group7.\n\nWe report a case of a malignant peripheral nerve sheath tumor of the thigh, without evidence of concomitant pulmonary metastases, which was found to have metastasized to the adrenal gland. This case raises the question if routine abdominal imaging should be performed along with pulmonary imaging in metastatic peripheral nerve sheath tumors.\n\n\nCase report\n\nA 40 year old woman with neurofibromatosis type 1 and no other significant past medical history or family history, presented for a hemorrhagic right thigh mass which had been enlarging over the past 3 months. The mass had previously been stable in size for a few years and was thought to be consistent with a neurofibroma. A CT scan of the extremities revealed a large mass within the posterior compartment of the right thigh, measuring 13.4 × 13.4 × 24.8 cm. In addition, an incidental 2 × 2cm left adrenal gland mass and 2 small lytic bone lesions (right 10th rib and right proximal femur) were also noted. Of note, a CT scan of the thorax, completed for staging purposes, did not reveal any suspicious pulmonary nodules or masses. An excisional biopsy of the thigh mass confirmed a high grade spindle cell sarcoma, negative for S100 protein, Desmin, CD31, AE1:AE3, HMB45, MelanA, SOX10 and TLE1. The lack of immunoreactivity for all performed markers for this tumor was most compatible with a malignant peripheral sheath tumor (Figure 1 and Figure 2). An MRI of the abdomen, to further evaluate the adrenal mass, revealed 2 small left adrenal gland lesions in the medial and lateral limbs. Given the known association of neurofibromatosis with pheochromocytoma, a biochemical workup showed elevated plasma free metanephrines, supporting the diagnosis of phaeochromocytoma. The patient underwent a laparoscopic adrenalectomy, with gross pathological exams revealing two tumor nodules and with a histological exam revealing an intermixed “collision” tumor involving pheochromocytoma and sarcoma, consistent with metastatic peripheral nerve sheath tumor. The patient refused radiation therapy and did not follow up with oncology.\n\nThe spindle-shaped nuclei have clumped chromatin. These features are compatible with a malignant peripheral nerve sheath tumor.\n\nOne population is composed of hypercellular malignant spindle cells with hyperchromatic nuclei (blue arrow) that are infiltrating the adjacent adrenal tissue. This is morphologically compatible with malignant peripheral nerve sheath tumor. The other population is composed of the nests of polygonal cells with abundant eosinophilic cytoplasm (green arrow), compatible with pheochromocytoma.\n\n\nDiscussion\n\nThe above case describes a patient with a malignant peripheral nerve sheath tumor (MPNST) of the thigh with pathologically confirmed metastases to the adrenal gland, yet without evidence of pulmonary metastases. The adrenal mass was found incidentally upon imaging of the thigh mass, but based on current NCCN guidelines6, a screening CT of the abdomen/pelvis (A/P) would not be indicated and thus could have potentially missed the presence of metastatic disease.\n\nTwo recent case series reached contradictory conclusions regarding the benefit of abdominal/pelvic CT screening. In the first case series, King, et al. evaluated 124 adult patients with sarcoma who underwent CT chest(C)/A/P imaging at their institution for staging and surveillance. Twenty (16%) of the patients had evidence of A/P metastasis, 7 on the initial scan and 13 on the surveillance8. Of note, six of the 20 patients (5% of the cohort) were found to have isolated A/P metastases without the development of pulmonary metastases during the study period. MPNST, specifically, made up 6% of the sarcomas evaluated and while no A/P metastases were found on screening, 2 patients had evidence on surveillance scans. Based on the finding that a wide variety of sarcoma subtypes were found to have extra-pulmonary disease, the authors conclude that A/P imaging should be included in the evaluation of all sarcoma subtypes. In contrast, Thompson et al. reviewed 140 patients of all ages who had a diagnosis of a malignant neoplasm of the upper or lower extremity and underwent screening and/or surveillance with a CT C/A/P9. Of those patients, 14 (10%) had evidence of abdominal/pelvic metastasis, with only 4 (2.9%) with evidence of isolated A/P disease. Additionally, of the 10 patients who developed metastases to both the chest and abdomen/pelvis, none developed evidence of disease in the abdomen/pelvis prior to the chest. Based on their results, the authors offer up the contrary opinion from King in that abdominopelvic imaging is not warranted in the sarcoma population.\n\nOur case adds a patient to the literature with a peripheral MPNST who was found to have an isolated adrenal metastasis without evidence of concomitant pulmonary disease. One striking feature of this case is the very large size of the primary tumor. Factors known to be associated with the development of metastases are tumor grade, tumor size, tumor depth, and certain histopathologies. Currently, though, guidelines only recommend CT of the chest for all patients and to consider CT A/P in patients with myxoid/round cell liposarcoma, epithelial sarcoma, angiosarcoma and leiomyosarcoma. Based on our case and the literature, we also would suggest adding screening CT A/P for large (>5cm), deep tumors of any histology.\n\n\nConclusions\n\nSoft tissue sarcomas are a heterogeneous group of tumors with a variety of prognoses. Deciding on a unified set of guidelines will be challenging, but given the clinical significance of finding metastatic disease, adding additional parameters (size and depth) to a more complete screening process would seem prudent.\n\n\nConsent\n\nWritten informed consent was obtained by the patient for publication of their clinical details. There are no potentially identifying images included in this paper.", "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\nClark MA, Fisher C, Judson I, et al.: Soft-tissue sarcomas in adults. N Engl J Med. 2005; 353(7): 701–11. PubMed Abstract \| Publisher Full Text\n\nKane JM 3rd: Surveillance strategies for patients following surgical resection of soft tissue sarcomas. Curr Opin Oncol. 2004; 16(4): 328–332. PubMed Abstract \| Publisher Full Text\n\nPisters PW, Leung DH, Woodruff J, et al.: Analysis of prognostic factors in 1,041 patients with localized soft tissue sarcomas of the extremities. J Clin Oncol. 1996; 14(5): 1679–1689. PubMed Abstract \| Publisher Full Text\n\nBillingsley KG, Lewis JJ, Leung DH, et al.: Multifactorial analysis of the survival of patients with distant metastasis arising from primary extremity sarcoma. Cancer. 1999; 85(2): 389–395. PubMed Abstract \| Publisher Full Text\n\nPotter DA, Glenn J, Kinsella T, et al.: Patterns of recurrence in patients with high-grade soft-tissue sarcomas. J Clin Oncol. 1985; 3(3): 353–366. PubMed Abstract \| Publisher Full Text\n\nvon Mehren M, Randall RL, Benjamin RS, et al.: Soft tissue sarcoma. Version 2. 2017, 2017. Reference Source\n\nESMO/European Sarcoma Network Working Group: Soft tissue and visceral sarcomas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2014; 25(Suppl 3): iii102–iii112. PubMed Abstract \| Publisher Full Text\n\nKing DM, Hackbarth DA, Kilian CM, et al.: Soft-tissue sarcoma metastases identified on abdomen and pelvis CT imaging. Clin Orthop Relat Res. 2009; 467(11): 2838–2844. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThompson MJ, Ross J, Domson G, et al.: Screening and surveillance CT abdomen/pelvis for metastases in patients with soft-tissue sarcoma of the extremity. Bone Joint Res. 2015; 4(3): 45–9. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "29129", "date": "20 Dec 2017", "name": "Ahmed Abu-Zaid", "expertise": [ "Reviewer Expertise Medical Oncology", "Surgical Oncology" ], "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\nWith interest, I read the article Shaines and Arora. Overall, the article reads very well, and should deserve acceptance following some minor/major changes to improve its quality and scientific soundness.\n1. Title: — You may want remove the phrase \"case report\". — Also, please change to \"an adrenal metastasis\".\n2. Abstract. — Please expand the abstract. — You may want to include a couple of sentences about definition of soft tissue sarcomas (STSs), incidence, biological behavior (with respect to abdominal/pelvis and distant metastasis) and percentage of MPNSTs. — You may also want to precisely mention the names of guidelines and dates. — All these recommended sentences above will highlight the importance of your case report. — The abstract says the STS of thigh was resected although this is not mentioned in the \"case report\" section. — Lastly, check if journal mandates a STRUCTURED format for the abstract section.\n\n3. Introduction: — It is recommended to add sentences that shed light on the most common sites of STSs and the percentage of MPNSTs among all STSs. — Mention the dates of guidelines.\n4. Case Report: — Add details about physical examination. — Add details about initial laboratory findings. — Add CT scans of the thigh mass (to highlight its large size) and adrenal mass (to highlight the collision tumor). — You add the MRI picture of the adrenal mass, too (or the CT scan). — Please do mention of the patient received surgery for the STS of the thigh as it is not clearly mentioned in this section.\n5. Discussion: — Since the two series presented in manuscript had contradictory conclusions, it is recommended (if available) to add additional data from one to two more series. — For reference (9), please add specific details about MPNSTs (if available). — Briefly mention how (why) MPNST are different from the myxoid liposarcoma, epithelial sarcoma, angiosarcoma etc in terms of the recommendation for CT A/P scanning. — please provide citations for the following sentence \"Factors known to be associated with the development of metastases are tumor grade, tumor size, tumor depth, and certain histopathologies\". 5. \"Based on our case and the literature, we also would suggest adding screening CT A/P for large (>5cm), deep tumors of any histology\". How depth was assessed in your case report.\n6. Conclusion: — Modify the conclusion so it will reflect the specific histopathological variant of MPNST (since it is not normally recommended by guidelines as opposed to the others).\n7. References: — Good\n8. English: — Minor English polishing.\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? Partly", "responses": [ { "c_id": "3543", "date": "09 Apr 2018", "name": "Shitij Arora", "role": "Author Response", "response": "We thank the reviewer for their excellent suggestions. Our response is included belowReviewers Response /1Abstract. — Please expand the abstract. — You may want to include a couple of sentences about definition of soft tissue sarcomas (STSs), incidence, biological behavior (with respect to abdominal/pelvis and distant metastasis) and percentage of MPNSTs. — You may also want to precisely mention the names of guidelines and dates. — All these recommended sentences above will highlight the importance of your case report. — The abstract says the STS of thigh was resected although this is not mentioned in the \"case report\" section. — Lastly, check if journal mandates a STRUCTURED format for the abstract section.Response- We included much of the above in an updated version of the introductionIntroduction: — It is recommended to add sentences that shed light on the most common sites of STSs and the percentage of MPNSTs among all STSs. — Mention the dates of guidelines.Response -UpdatedCase Report: — Add details about physical examination. — Add details about initial laboratory findings. — Add CT scans of the thigh mass (to highlight its large size) and adrenal mass (to highlight the collision tumor). — You add the MRI picture of the adrenal mass, too (or the CT scan). — Please do mention of the patient received surgery for the STS of the thigh as it is not clearly mentioned in this section.Response- Updated much of this.We have attached an image of the thigh mass with this response instead – please click here to see this.Discussion: — Since the two series presented in manuscript had contradictory conclusions, it is recommended (if available) to add additional data from one to two more series. — For reference (9), please add specific details about MPNSTs (if available). — Briefly mention how (why) MPNST are different from the myxoid liposarcoma, epithelial sarcoma, angiosarcoma etc in terms of the recommendation for CT A/P scanning. — please provide citations for the following sentence \"Factors known to be associated with the development of metastases are tumor grade, tumor size, tumor depth, and certain histopathologies\". 5. \"Based on our case and the literature, we also would suggest adding screening CT A/P for large (>5cm), deep tumors of any histology\". How depth was assessed in your case report.Response -Updated much of thisThese are the only 2 case series we could find that address this issue of A/P imagingwe removed “depth” from that last sentence as it really does appear that size if what mattersConclusion: — Modify the conclusion so it will reflect the specific histopathological variant of MPNST (since it is not normally recommended by guidelines as opposed to the others).Response- Wepurposely left this vague because this this was only one case of MPNST and there are so many variants of soft tissue sarcoma. I thought it would be more generalizable to state tumor size, in general, should be thought of as important when it comes to screening imaging since it is noted as a risk factor for all STS." } ] }, { "id": "29117", "date": "08 Jan 2018", "name": "Shweta Gera", "expertise": [ "Reviewer Expertise Pathology", "oncology" ], "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 case report describes an interesting finding but it needs major revision. Some of the changes suggested as below-\n\nIt describes an NF type-1 patient with MPNST with metastasis to the adrenal gland. The patients with NF type-1 have increased risk for pheochromocytoma. Likely the patient had undiagnosed pheochromocytoma (was the patient hypertensive?) and the patient developed metastasis to pheochromocytoma. So it describes more of cancer-to-cancer metastasis than collision tumor. Add more references with respect to cancer-to-cancer metastasis. Collision tumor is defined as two distinct tumors developing in juxtaposition to one another without areas of intermingling. In the current case, as in Figure 2, there is an intermingling of both components- so again it is not a collision tumor. The title needs to be changed and make it apt.\n\nSpelling errors like prognoses (mentioned in conclusion). There is nothing called epithelial sarcoma, the correct terminology is epithelioid sarcoma. MPNST stands for malignant peripheral nerve sheath tumor so no need for peripheral MPNST (mentioned in the first line of the last paragraph of discussion). Please check for grammatical errors as well.\n\nAbstract- talks about guidelines for staging peripheral nerve sheath tumors, which sounds like it was a benign tumor. So use MPNST instead of just peripheral nerve sheath tumor. Abstract and discussion mention isolated adrenal metastasis, which is not true; she had both bone and adrenal metastasis. There is mention that- \"She underwent primary and adrenal metastasis resection.\" consider revising it to primary tumor resection and adrenalectomy for metastasis.\n\nExpand on physical exam of thigh mass- size, tenderness, consistency etc. Elaborate on findings of MRI with details of imaging findings of adrenal metastasis.\n\nExpand on gross findings of thigh mass and adrenal nodules- eg. size, color, infiltrative or well-circumscribed etc. Did both the adrenal nodules have similar histological findings? Were any immunostains done to confirm pheochromocytoma component?\n\nRef 9 talks about patients with malignant neoplasm of the upper or lower extremity. There is no mention of that those were sarcomas. Check for that.\n\nExpand on abstract- add more about the significance of the findings of the case; expand on introduction and discussion- add more details for sarcoma, development of MPNST in association to NF-1, adrenal metastasis, management guidelines, more references for the recommendation for screening CT A/P for sarcomas. In the discussion, authors have suggested adding screening CT A/P for large (>5cm), deep tumors of any histology- expand more on this by adding more references.\n\nAdd limitations of the study, the major limitation being small sample size.\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": [ { "c_id": "3542", "date": "09 Apr 2018", "name": "Shitij Arora", "role": "Author Response", "response": "We thank you for the review and the excellent suggestions. Our response is included belowt describes an NF type-1 patient with MPNST with metastasis to the adrenal gland. The patients with NF type-1 have increased risk for pheochromocytoma. Likely the patient had undiagnosed pheochromocytoma (was the patient hypertensive?) and the patient developed metastasis to pheochromocytoma. So it describes more of cancer-to-cancer metastasis than collision tumor. Add more references with respect to cancer-to-cancer metastasis. Collision tumor is defined as two distinct tumors developing in juxtaposition to one another without areas of intermingling. In the current case, as in Figure 2, there is an intermingling of both components- so again it is not a collision tumor. The title needs to be changed and make it apt.Response - term collision tumor is removed.Spelling errors like prognoses (mentioned in conclusion). There is nothing called epithelial sarcoma, the correct terminology is epithelioid sarcoma. MPNST stands for malignant peripheral nerve sheath tumor so no need for peripheral MPNST (mentioned in the first line of the last paragraph of discussion). Please check for grammatical errors as well. Response - correctedAbstract- talks about guidelines for staging peripheral nerve sheath tumors, which sounds like it was a benign tumor. So use MPNST instead of just peripheral nerve sheath tumor. Abstract and discussion mention isolated adrenal metastasis, which is not true; she had both bone and adrenal metastasis. There is mention that- \"She underwent primary and adrenal metastasis resection.\" consider revising it to primary tumor resection and adrenalectomy for metastasis. Response - correctedExpand on physical exam of thigh mass- size, tenderness, consistency etc. Elaborate on findings of MRI with details of imaging findings of adrenal metastasis. Response- correctedExpand on gross findings of thigh mass and adrenal nodules- eg. size, color, infiltrative or well-circumscribed etc. Did both the adrenal nodules have similar histological findings? Were any immunostains done to confirm pheochromocytoma component? Response- immunostains are included in the findings described dd limitations of the study, the major limitation being small sample size.Response- We intend to publish this as a case report" } ] } ]	1	https://f1000research.com/articles/6-1964
https://f1000research.com/articles/7-174/v1	12 Feb 18	{ "type": "Research Article", "title": "Intraocular pressure elevation precedes a phagocytosis decline in a model of pigmentary glaucoma", "authors": [ "Yalong Dang", "Susannah Waxman", "Chao Wang", "Priyal Shah", "Ralitsa T. Loewen", "Nils A. Loewen", "Yalong Dang", "Susannah Waxman", "Chao Wang", "Priyal Shah", "Ralitsa T. Loewen" ], "abstract": "Background: Outflow regulation and phagocytosis are key functions of the trabecular meshwork (TM), but it is not clear how the two are related in secondary open angle glaucomas characterized by an increased particle load. We hypothesized that diminished TM phagocytosis is not the primary cause of early ocular hypertension and recreated pigment dispersion in a porcine ex vivo model. Methods: Sixteen porcine anterior chamber cultures received a continuous infusion of pigment granules (Pg), while 16 additional anterior chambers served as controls (C). Pressure transducers recorded the intraocular pressure (IOP). The phagocytic capacity of the trabecular meshwork was determined by fluorescent microspheres. Results: The baseline IOPs in Pg and C were similar (P=0.82). A significant IOP elevation occurred in Pg at 48, 120, and 180 hours (all P<0.01, compared to baseline). The pigment did not cause a reduction in TM phagocytosis at 48 hours, when the earliest IOP elevation occurred, but at 120 hours onward (P=0.001 compared to C). This reduction did not result in an additional IOP increase at 120 or 180 hours compared to the first IOP elevation at 48 hours (P>0.05). Conclusions: In this porcine model of pigmentary glaucoma, an IOP elevation occurs much earlier than when phagocytosis fails, suggesting that two separate mechanisms might be at work.", "keywords": [ "Pigment dispersion glaucoma", "aqueous outflow", "trabecular meshwork", "intraocular pressure", "phagocytosis" ], "content": "Introduction\n\nThe conventional outflow is guarded by the trabecular meshwork (TM), a complex three dimensional, layered tissue that contains variable amounts of extracellular matrix (ECM)1. The aqueous passes into Schlemm's canal by paracytosis or giant vacuoles2. Failure to maintain a normal cytoskeleton and homeostasis of aqueous outflow can cause ocular hypertension1. For instance, pigment dispersion3 and corticosteroids can alter the actin cytoskeleton and cause TM cell contraction resulting in an elevation of intraocular pressure (IOP)3,4. Conversely, relaxing the cytoskeleton, for instance by using a Rho kinase inhibitor, can reverse these effects5,6.\n\nPhagocytosis of debris is another key function of TM cells2. However, its direct and short-term effects on IOP regulation remain poorly understood1. Chronic exposure to pigment7, erythrocyte-derived ghost cells8, inflammatory cells9, photoreceptor outer segments10, lens and pseudoexfoliation material11,12 can lead to secondary glaucomas.\n\nWe recently developed an ex vivo pigmentary glaucoma (PG) model that recreates the IOP elevation, stress fiber formation, and phagocytosis reduction characteristics of human PG3. A gene expression analysis indicated an activation of the RhoA signaling pathway, and a downstream effect of tight junction formation negatively regulated by RhoA-mediated actin cytoskeletal reorganization3. In the current study, we hypothesized that ocular hypertension is the result of a reorganization of the actin cytoskeleton and occurs before phagocytosis declines.\n\n\nMethods\n\nThis study was conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Because no live vertebrate animals were used and pig eyes were acquired from a local abattoir (Thoma Meat Market, Saxonburg, PA), no Institutional Animal Care and Use approval was required.\n\nThirty-two porcine eyes were cultured within 2 hours of enucleation. Extraocular tissues were removed, and the eyes were decontaminated with 5% povidone-iodine solution (CAT# 3955-16, United States Pharmacopeia, Rockville, MD) for two minutes and washed three times in phosphate buffered saline (PBS). Posterior segments, lenses, and irises were removed and the anterior segments with intact TM mounted in the perfusion system as previously described3,13,14. We used the same method to generate pigment granules as recently described in a model of pigmentary glaucoma (PG)3. Briefly, pigment granules were produced by subjecting the iris to freeze-thaw and resuspension washing before dilution of the stock to a final concentration of 1.67×107 particles/ml. Eyes in the pigment dispersion group were continuously perfused with pigment added to the culture medium for up to 180 hours (Pg) and compared to controls (C). The perfusate consisted of Dulbecco's modified Eagle media (DMEM, SH30284, HyClone, GE Healthcare, UK) supplemented with 1% FBS and 1% antibiotics (15240062, Thermo Fisher Scientific, Waltham, MA) at a constant rate of 3 µl/min using a microinfusion pump (PHD 22/2000; Harvard Apparatus, Holliston, MA). IOP was measured intracamerally by a pressure transducer (SP844; MEMSCAP, Skoppum, Norway) and recorded at two-minute intervals (LabChart, ADInstruments, Colorado Springs, CO). Baseline IOPs were obtained after IOP stabilization for 48 hours.\n\nThe in situ TM phagocytosis was measured using an epifluorescence microscope after microsphere perfusion. In brief, a suspension of 0.5 μm carboxylate-modified yellow-green fluorescent microspheres15 (CAT# F8813, Thermo Fisher, Waltham, MA) at 5×108 particles/ml was added to the perfusate at 48, 120, and 180 hours and perfused for 24 hours. The eyes were removed from their perfusion dishes, washed three times with pre-warmed PBS, secured again in the perfusion dishes, and placed upside down for imaging. The TM, visualized from the underside of the transparent perfusion dish, was photographed and measured by acquiring the images with a camera and epifluorescence equipped dissecting fluorescence microscope (SZX16, Olympus, Tokyo, Japan) at a 680×510 pixel resolution and a 200 ms exposure. The mean fluorescence intensity was quantified by ImageJ (Version 1.50i, NIH) as previously described16 at 48, 120, and 180 hours by measuring the fluorescence intensity in the TM.\n\nTo validate that the microspheres were phagocytosed by TM cells, the TM was dissected and digested with collagenase type IA (C9891, Sigma Aldrich, St. Louis, MO) at 2mg/ml and 1% FBS for 30 min at room temperature. The cells were filtered with a 70-micron cell strainer and resuspended in 0.5 ml of PBS. The percentage of TM cells that had ingested fluorescent microspheres was determined using flow cytometry.\n\nTo get a more accurate visualization of the phagocytosed microbeads, we used confocal microscopy. TM cells were seeded into the wells of a six-well plate and fixed with 4% PFA. The cell membranes were labeled with Lycopersicon esculentum agglutinin (TL; Texas red-conjugated; #TL-1176, Vector Laboratories, Inc., Burlingame, CA) at room temperature for 1 hour. The cell nuclei were counterstained with DAPI (D1306, Thermo Fisher Scientific, Waltham, MA). Photos and 3D videos were taken using an upright laser scanning confocal at 400x magnification (BX61, Olympus, Tokyo, Japan).\n\nAfter the TM phagocytosis assay, the anterior segments were fixed with 4% PFA for 24 hours, washed three times with PBS, dehydrated in 70% ethanol, and embedded in paraffin. Sections were cut to a thickness of 5 μm and stained with hematoxylin and eosin (H&E).\n\nData were presented as the mean ± standard error and analyzed by PASW Statistics 18 (SPSS Inc., Chicago, IL). The baseline IOP was compared to the other time points of the same eye using a paired t-test. Other quantitative data were analyzed by one-way ANOVA. A p value ≤ 0.05 was considered statistically significant.\n\n\nResults\n\nIn H&E stained tissue sections, normal TM (Figure 1A) presented as a sparsely pigmented (red arrowheads), multilayered, porous tissue with Schlemm’s canal-like segments within the aqueous plexus at the outer layer (black arrows). Pigment granules were seen phagocytosed by trabecular meshwork cells, particularly in the uveal TM, at 48, 120, and 180 hours (Figure 1B, C and D) but were not dense enough to physically obstruct any part of the conventional outflow system.\n\nNormal trabecular meshwork (TM) (A) was a multilayer, strainer-like structure with few pigment deposits (red arrowheads). Ex vivo perfusion with pigment granules at 1.67×107/ml caused significant TM pigmentation at 48 hours (B), 120 hours (C) and 180 hours (D). No apparent occlusion of the outflow tract was found.\n\nBaseline IOP in Pg was comparable to C (12.2±0.9 mmHg vs. 11.9±0.9 mmHg, P=0.82). Pigment dispersion caused a significant IOP elevation at 48, 120, and 180 hours (19.5±1.4 mmHg, 20.2±1.4 mmHg and 22.8±0.8 mmHg, P=0.001, P<0.001 and P=0.002, compared to baseline) while IOPs in C remained steady (13.1±1.1 mmHg, 12.0±0.9 mmHg and 14.0±1.5 mmHg, all p values >0.05, compared to baseline) (Figure 2A).\n\nBaseline IOPs in the pigment group (n=16) and the control (n=16) are comparable (12.2±0.9 mmHg vs. 11.9±0.9 mmHg, P=0.82). Pigment caused a significant IOP elevation at 48 hours and onward (all P<0.05) while the IOP in the control group showed no significant difference to baseline at any time point (all P>0.05) when compared to the baseline (A). TM phagocytosis was visualized in situ. The mean fluorescence intensity in the TM region was quantified by NIH ImageJ. TM phagocytosis in the pigment group was comparable to the control at 48 hours (P=0.723), (Bi–ii) but showed sharp decreases at 120 hours (Biii–iv) and 180 hours (P=0.001 and P=0.026, respectively) (Bv–vi).\n\nBy inverting the perfusion dishes and washing away the microspheres in the intertrabecular spaces, the TM phagocytosis was visualized and quantified under an upright dissecting fluorescence microscope. Pigment did not cause any change of phagocytosis during early ocular hypertension at 48 hours (Figure 2Bi-ii, 96.3±5.0% compared to the control, P=0.723), but did cause a reduction at the later phases of 120 hours (Figure 2Biii-iv, 58.3±2.3%, P=0.001) and 180 hours (Figure2Bv-vi, 62.5±5.1%, P=0.026). However, the declining phagocytosis did not result in further elevation of IOP at 120 and 180 hours compared to the initial IOP elevation at 48 hours (20.2±1.4 mmHg and 22.8±0.8 mmHg versus 19.5±1.4 mmHg, both P>0.05).\n\nThe microsphere ingestion by TM cells was further assessed by flow cytometry and confocal microscopy. 28.1% of TM cells had phagocytosed microbeads in a normal perfusion eye (Figure 3A) and the confocal microscopy confirmed them as being located within the cells (Figure 3B) aided by tomato lectin-stained cell membranes and DAPI-stained nuclei. Confocal imaging showed clusters of green fluorescent microspheres within the intracellular space with no microspheres in the intercellular space. The 3D video also suggested the microspheres were in fact phagocytized and not merely on top of or below them since the microbeads were in the same z plane as the cells (Supplementary Video 1).\n\nTo further confirm that microspheres were phagocytosed, we digested a normal sample TM tissue into single cell suspension and sent for flow cytometry. The results suggested that 28.1% of the TM cells were actively phagocytic (A). We then seeded these cells into a six well plate to form monolayer. After labeling them with tomato lectin, the confocal imaging showed that clusters of green fluorescent microspheres were located in the intracellular but not in the intercellular space (B).\n\n\nDiscussion\n\nPhagocytosis is a defining feature of TM cells17 and plays a central but poorly understood role in the pathogenesis of several types of secondary glaucoma that include pigment, erythrocytes and ghost cells, inflammatory cells, photoreceptor outer segments, lens and pseudoexfoliation material3,18,19. Although TM phagocytosis can remove particles from the aqueous humor20, the direct and short-term effects on outflow regulation remain insufficiently explained1. In this study, we measured IOP and TM phagocytic activity in the presence of pigment granules at different time points and found IOP was significantly elevated as early as 48 hours after exposure to pigment granules. This was contrasted by a phagocytic activity in Pg not different from C before the decrease at 120 and 180 hours. A worsening decline of TM phagocytosis at 120 and 180 hours did not result in a further increase of IOP. This suggests that reduction in phagocytosis is a downstream and secondary effect of actin cytoskeletal reorganization.\n\nPigment treatment has previously been shown to cause ocular hypertension in part by reorganizing the TM actin cytoskeleton and not by physical obstruction of the outflow tract7,21. We have recently reported that long, thick, and continuous TM actin bundles emerge as early as 24 hours after pigment exposure3 and replicate this observation in the present study. Histological characteristics of pigment dispersion in porcine eyes matched those seen in samples from pigmentary glaucoma patients21–23 showing that pigment particles were taken up by TM cells.\n\nIn summary, the results indicate the IOP elevation caused by pigment dispersion is not the direct result of a physical obstruction of outflow or a chronically overwhelmed phagocytosis. The reduction in phagocytosis considerably lags the evolving hypertension supporting the notion that these cytoskeletal changes occur early on and are separate from the impact of pigment on canonical phagocytosis pathways3.\n\n\nData availability\n\nAll the raw data generated or analyzed in this study are included in following datasets.\n\nDataset 1. Raw unedited images of Figure 1. They are representative of 17 slides for histology. 10.5256/f1000research.13797.d19208824\n\nDataset 2. Raw unedited images of Figure 2B. They are representative of 31 pictures for phagocytosis measurement. 10.5256/f1000research.13797.d19208925\n\nDataset 3. Raw unedited images of Figure 3B. 10.5256/f1000research.13797.d19209026\n\nDataset 4. The FACS output file for Figure 3A. 10.5256/f1000research.13797.d19209127\n\nDataset 5. The raw IOP and phagocytosis measurements at all time points. 10.5256/f1000research.13797.d19209228", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nNIH CORE Grant P30 EY08098 to the Department of Ophthalmology, from the Eye and Ear Foundation of Pittsburgh, and from an unrestricted grant from Research to Prevent Blindness, New York, NY; National Eye Institute K08EY022737 (NAL); Initiative to Cure Glaucoma of the Eye and Ear Foundation of Pittsburgh (NAL); Research to Prevent Blindness, Departmental Grant (NAL); the Wiegand Fellowship of the Eye and Ear Foundation (YD); an unrestricted grant from the Third Xiangya Hospital of Central South University for studying at the University of Pittsburgh (CW).\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 Video 1. Visualization of microsphere ingestion by a 3D reconstruction with confocal microscopy. We took a series of z-stack confocal microscopy images to reconstruct a 3D video, showing that the fluorescent microspheres were neither on the top nor below, but phagocytized by the TM cells.\n\nClick here to access the data.\n\n\nReferences\n\nLlobet A, Gasull X, Gual A: Understanding trabecular meshwork physiology: a key to the control of intraocular pressure? News Physiol Sci. 2003; 18(5): 205–9. PubMed Abstract \| Publisher Full Text\n\nTamm ER: The trabecular meshwork outflow pathways: structural and functional aspects. Exp Eye Res. 2009; 88(4): 648–55. PubMed Abstract \| Publisher Full Text\n\nDang Y, Waxman S, Wang C, et al.: Trabecular Meshwork Failure In A Model Of Pigmentary Glaucoma [Internet]. bioRxiv. 2017; 118448, [cited 2017 Apr 21]. Reference Source\n\nPattabiraman PP, Rao PV: Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells. Am J Physiol Cell Physiol. 2010; 298(3): C749–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTanihara H, Inoue T, Yamamoto T, et al.: Intra-ocular pressure-lowering effects of a Rho kinase inhibitor, ripasudil (K-115), over 24 hours in primary open-angle glaucoma and ocular hypertension: a randomized, open-label, crossover study. Acta Ophthalmol. 2015; 93(4): e254–60. PubMed Abstract \| Publisher Full Text\n\nTanihara H, Inoue T, Yamamoto T, et al.: Phase 1 clinical trials of a selective Rho kinase inhibitor, K-115. JAMA Ophthalmol. 2013; 131(10): 1288–95. PubMed Abstract \| Publisher Full Text\n\nEpstein DL, Freddo TF, Anderson PJ, et al.: Experimental obstruction to aqueous outflow by pigment particles in living monkeys. Invest Ophthalmol Vis Sci. 1986; 27(3): 387–95. PubMed Abstract\n\nCampbell DG, Simmons RJ, Grant WM: Ghost cells as a cause of glaucoma. Am J Ophthalmol. 1976; 81(4): 441–50. PubMed Abstract \| Publisher Full Text\n\nMoorthy RS, Mermoud A, Baerveldt G, et al.: Glaucoma associated with uveitis. Surv Ophthalmol. 1997; 41(5): 361–94. PubMed Abstract \| Publisher Full Text\n\nCallender D, Jay JL, Barrie T: Schwartz-Matsuo syndrome: atypical presentation as acute open angle glaucoma. Br J Ophthalmol. 1997; 81(7): 608–608. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee RK: The molecular pathophysiology of pseudoexfoliation glaucoma. Curr Opin Ophthalmol. 2008; 19(2): 95–101. PubMed Abstract \| Publisher Full Text\n\nRitch R, Schlötzer-Schrehardt U, Konstas AG: Why is glaucoma associated with exfoliation syndrome? Prog Retin Eye Res. 2003; 22(3): 253–75. PubMed Abstract \| Publisher Full Text\n\nLoewen RT, Roy P, Park DB, et al.: A Porcine Anterior Segment Perfusion and Transduction Model With Direct Visualization of the Trabecular Meshwork. Invest Ophthalmol Vis Sci. 2016; 57(3): 1338–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDang Y, Waxman S, Wang C, et al.: Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model. Peer J Preprints. 2017; 5: [cited 2017 Jan 24]. Report No.: e2736v1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoewen RT, Brown EN, Scott G, et al.: Quantification of Focal Outflow Enhancement Using Differential Canalograms. Invest Ophthalmol Vis Sci. 2016; 57(6): 2831–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDang Y, Waxman S, Wang C, et al.: Rapid learning curve assessment in an ex vivo training system for microincisional glaucoma surgery. PeerJ Preprints. 2017; [cited 2017 Jan 25]. Report No. 5: e2745v1. Publisher Full Text\n\nBuller C, Johnson DH, Tschumper RC: Human trabecular meshwork phagocytosis. Observations in an organ culture system. Invest Ophthalmol Vis Sci. 1990; 31(10): 2156–63. PubMed Abstract\n\nLiton PB, Lin Y, Gonzalez P, et al.: Potential role of lysosomal dysfunction in the pathogenesis of primary open angle glaucoma. Autophagy. 2009; 5(1): 122–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang X, Ognibene CM, Clark AF, et al.: Dexamethasone inhibition of trabecular meshwork cell phagocytosis and its modulation by glucocorticoid receptor beta. Exp Eye Res. 2007; 84(2): 275–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbu-Hassan DW, Acott TS, Kelley MJ: The Trabecular Meshwork: A Basic Review of Form and Function. J Ocul Biol. 2014; 2(1): pii: http://fulltextarticles.avensonline.org/JOCB-2334-2838-02-0017.html. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGottanka J, Johnson DH, Grehn F, et al.: Histologic findings in pigment dispersion syndrome and pigmentary glaucoma. J Glaucoma. 2006; 15(2): 142–51. PubMed Abstract \| Publisher Full Text\n\nKupfer C, Kuwabara T, Kaiser-Kupfer M: The histopathology of pigmentary dispersion syndrome with glaucoma. Am J Ophthalmol. 1975; 80(5): 857–62. PubMed Abstract \| Publisher Full Text\n\nAlvarado JA, Murphy CG: Outflow obstruction in pigmentary and primary open angle glaucoma. Arch Ophthalmol. 1992; 110(12): 1769–78. PubMed Abstract \| Publisher Full Text\n\nDang Y, Waxman S, Wang C, et al.: Dataset 1 in: Intraocular pressure elevation precedes a phagocytosis decline in a model of pigmentary glaucoma. F1000Research. 2018. Data Source\n\nDang Y, Waxman S, Wang C, et al.: Dataset 2 in: Intraocular pressure elevation precedes a phagocytosis decline in a model of pigmentary glaucoma. F1000Research. 2018. Data Source\n\nDang Y, Waxman S, Wang C, et al.: Dataset 3 in: Intraocular pressure elevation precedes a phagocytosis decline in a model of pigmentary glaucoma. F1000Research. 2018. Data Source\n\nDang Y, Waxman S, Wang C, et al.: Dataset 4 in: Intraocular pressure elevation precedes a phagocytosis decline in a model of pigmentary glaucoma. F1000Research. 2018. Data Source\n\nDang Y, Waxman S, Wang C, et al.: Dataset 5 in: Intraocular pressure elevation precedes a phagocytosis decline in a model of pigmentary glaucoma. F1000Research. 2018. Data Source" }	[ { "id": "32109", "date": "26 Mar 2018", "name": "Sergio C Saccà", "expertise": [ "Reviewer Expertise Glaucoma pathogenesis and Molecular 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\nI read the article titled: Intraocular pressure elevation precedes a phagocytosis decline in a model of pigmentary glaucoma by Dang et al. The title of the article is very stimulating but the content has several things that should be corrected. The authors state that in the conventional way of outflow the aqueous passes into Schlemm's canal by paracytosis or giant vacuoles. This is a partial view that does not correspond to the truth. I invite the authors to read Saccà et al. The Outflow Pathway: A Tissue With Morphological and Functional Unity1. Then the effects of the inhibitors of the Rho kinase inhibitor may have the ability to modify the metabolism of endothelial cells in the node in which the authors think it is open to question. These drugs have the ability to block TM motility by exposing more cells to aqueous humor. This is the reason why we initially witness a decline in IOP then this worsens because the exposed cells are blocked and are more exposed to apoptosis (to read Saccà et al. from DNA damage to functional changes of the trabecular meshwork in aging and glaucoma2). Also the hypothesis formulated by the authors to explain the pigmentary glaucoma is partial in the Trabecolato the so-called pores have a very low meaning and above all the pigment does not obstruct anything. We do not work like sinks - I am amazed that today we can still believe that TM is a porous tissue, and above all, how it is possible to think that outflow is a passive phenomenon. In pigmentary glaucoma there are 2 fundamental pathogenic moments. The first concerns the back sheet of the iris, where the pigment comes from (a defect that involves the loss of the pigment itself) the second point concerns autophagy which is not able to ensure adequate cell homeostasis. So these cells first enter suffering and then die and hence glaucoma is why the cells do not work, not because it obstructs something. There are cells, not tubes. The cytoskeleton in all this has little to do with it: pigment granules are only a metabolic burden that in the case of pigment dispersal syndrome is absorbed by a functioning autophagy, while in pigment glaucoma, autophagy does not work. Finally, I remember that the changes in the cytoskeleton occur in all types of glaucoma. I do not believe that this article can be indexed in this form and needs a remake of both the introduction and the discussion and the results should be reviewed in the light of a more modern interpretation of the physio-pathogenical events.\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? 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? No", "responses": [ { "c_id": "3570", "date": "09 Apr 2018", "name": "Nils Loewen", "role": "Author Response", "response": "Reviewer 1: I read the article titled: Intraocular pressure elevation precedes a phagocytosis decline in a model of pigmentary glaucoma by Dang et al. The title of the article is very stimulating but the content has several things that should be corrected. Authors: We thank the reviewer, Professor Saccà, for having taken the time to carefully review our manuscript. We hope that we are able to address all of them with extensive manuscript changes as requested. Reviewer 1: 1)The authors state that in the conventional way of outflow the aqueous passes into Schlemm's canal by paracytosis or giant vacuoles. This is a partial view that does not correspond to the truth. I invite the authors to read Saccà et al. The Outflow Pathway: A Tissue With Morphological and Functional Unity1. Authors: Thank you for bringing this review article to our attention. We are pleased to use this publication and your other article as additional references. We have modified the Introduction to include more comprehensive details about conventional outflow better aligned with Saccà et al. We would like to change the less common phrase “paracytosis” with “paracellular passage through pores” to distinguish it from the active process of pathogen intrusion through epithelia e.g. by haemophilus.1 Overall, it appears our manuscript matches Saccà et al well who state that the main mechanisms of aqueous outflow are “a paracellular route [...] and a transcellular pathway”. We have added to the Introduction the description of a transcellular pathway through intracellular pores2 with inducible pore density that depends on outflow demand3. In the reworded Introduction we now state: “The aqueous passes from the anterior chamber into Schlemm's canal (SC) by entering first the uveal TM (UTM), the corneoscleral TM (CTM) and finally the juxtacanalicular TM (JCT)4. The JCT contains proteoglycans and hyaluronans and presents the aqueous humor with an increasingly tighter fluid passageway towards the SC in a process referred to as funneling5. The aqueous eventually passes the inner wall of SC endothelium mainly by two different mechanisms, a paracellular route in between endothelial cells and a transcellular route6 consisting of intracellular pores and giant vacuoles that are time and pressure dependent7–9. Failure of the TM and the inner wall of SC endothelial cells to maintain homeostasis and a normal cytoskeleton and can cause ocular hypertension10. For instance, pigment dispersion11 and corticosteroids can increase and contract actin stress fibers and result in an elevation of the intraocular pressure (IOP)11,12. Conversely, relaxing the cytoskeleton with Rho kinase inhibitors can reverse these effects13,14. Phagocytosis of debris is another function of TM cells8. A chronic phagocytosis demand in the form of pigment15, erythrocyte-derived ghost cells16, inflammatory cells17, photoreceptor outer segments18, lens and pseudoexfoliation material19,20 can all lead to secondary glaucomas even though the amount of material itself is unlikely to cause a physical outflow obstruction. Although these glaucomas make for a sizable fraction of open angle glaucomas, it was difficult to study the cellular mechanism that leads to an IOP elevation.” 2) Then the effects of the inhibitors of the Rho kinase inhibitor may have the ability to modify the metabolism of endothelial cells in the node in which the authors think it is open to question. These drugs have the ability to block TM motility by exposing more cells to aqueous humor. This is the reason why we initially witness a decline in IOP then this worsens because the exposed cells are blocked and are more exposed to apoptosis (to read Saccà et al. from DNA damage to functional changes of the trabecular meshwork in aging and glaucoma2). Authors: We completely agree with the Reviewer. We would like to point out that - consistent with Sacca et al - we wrote that “we hypothesized that ocular hypertension is the result of a reorganization of the actin cytoskeleton and occurs before phagocytosis declines.” We did not intend to describe outflow through the TM as sink-like. We hope the expanded Introduction makes it more obvious that this is a paracellular and transcellular process. In addition, we specifically stated before that “material itself is unlikely to cause a physical outflow obstruction” (Introduction), that granules “were not dense enough to physically obstruct any part of the conventional outflow system” (Results) and discuss that this had already been shown before: “Pigment treatment has previously been shown to cause ocular hypertension in part by reorganizing the TM actin cytoskeleton and not by physical obstruction of the outflow tract15,21.” (Discussion). We now clarify in the Discussion that “Past investigations suggested that the outflow obstruction was, in fact, physical15,22 but newer studies indicated that ocular hypertension is in part caused indirectly by reorganization of the TM actin cytoskeleton23. Consistent with Zhou et al.23, we recently reported that long, thick, and continuous TM actin bundles emerge as early as 24 hours after pigment exposure11 and replicate this observation in the present study.” 3) Also the hypothesis formulated by the authors to explain the pigmentary glaucoma is partial in the Trabecolato the so-called pores have a very low meaning and above all the pigment does not obstruct anything. We do not work like sinks - I am amazed that today we can still believe that TM is a porous tissue, and above all, how it is possible to think that outflow is a passive phenomenon. Authors: We did not state anything about an outflow obstruction or a location in our hypothesis. We wrote, “In the current study, we hypothesized that ocular hypertension is the result of a reorganization of the actin cytoskeleton and occurs before phagocytosis declines.” As we detail in our Response to 3), we never wrote that the TM functions as a sink. On the contrary, we elaborate multiple times that there is no physical obstruction as detailed in our Response to 2). Please see our response above where we hope to clarify this point in the rewritten manuscript. 4) In pigmentary glaucoma there are 2 fundamental pathogenic moments. The first concerns the back sheet of the iris, where the pigment comes from (a defect that involves the loss of the pigment itself) the second point concerns autophagy which is not able to ensure adequate cell homeostasis. So these cells first enter suffering and then die and hence glaucoma is why the cells do not work, not because it obstructs something. There are cells, not tubes. Authors: We completely agree as detailed in 1)-3) and have expanded our manuscript to make this more obvious. 5) The cytoskeleton in all this has little to do with it: pigment granules are only a metabolic burden that in the case of pigment dispersal syndrome is absorbed by a functioning autophagy, while in pigment glaucoma, autophagy does not work. Finally, I remember that the changes in the cytoskeleton occur in all types of glaucoma. I do not believe that this article can be indexed in this form and needs a remake of both the introduction and the discussion and the results should be reviewed in the light of a more modern interpretation of the physio-pathogenical events. Authors: We are unsure why the Reviewer states that cytoskeletal changes have nothing to do with an IOP elevation after a phagocytosis challenge. This is rather well established: Zhou et al found that 4 h after phagocytosis, the cytoskeletal structure in trabecular meshwork cells was disrupted23. As we detail in the Introduction “pigment dispersion11 and corticosteroids can increase and contract actin stress fibers and result in an elevation of the intraocular pressure (IOP)11,12. Conversely, relaxing the cytoskeleton with Rho kinase inhibitors can reverse these effects13,14.” The new finding described in this manuscript is that the IOP rises when the cytoskeleton changes occur as a result form pigment exposure but before phagocytosis starts to fail. This has not been observed before because an appropriate ex vivo anterior segment model of pigment dispersion did not exist. Our recent studies11,24,25 show that ex vivo perfused pig eyes experience reduced outflow in response to continuous exposure to pigment at a concentration far lower (10,000-fold) than that used in previous bolus experiments26 and that severe cytoskeletal changes are associated with this. Conversely, we show that rho-kinase inhibitors normalize the cytoskeleton and the phagocytosis24. TM regulates aqueous outflow by changing its cytoskeleton, stiffness, cell adhesion, migration, contraction, and phagocytosis10,27, in which ROCK signaling plays a central role28,29. There is a close interaction between actin cytoskeleton reorganization, cell or extracellular matrix stiffness and aqueous outflow facility30,31. Only a small proportion of stress fiber formation was found in the normal TM culture while pigment exposure increased it by 2.06-fold 11. Lastly, we caution not to confuse TM cell phagocytosis of pigmented debris with autophagy. To address the Reviewer’s concern we have added to the Discussion his own reference32 and state that “An increased pigmentary debris may interfere with many intracellular functions, including important autophagy functions that can cause a gradual deterioration of TM cell function32,33.” References used by Reviewer 1 1. Saccà SC, Gandolfi S, Bagnis A, Manni G, Damonte G, Traverso CE, Izzotti A: The Outflow Pathway: A Tissue With Morphological and Functional Unity.J Cell Physiol. 2016; 231 (9): 1876-93 PubMed Abstract \| Publisher Full Text 2. Saccà SC, Gandolfi S, Bagnis A, Manni G, Damonte G, Traverso CE, Izzotti A: From DNA damage to functional changes of the trabecular meshwork in aging and glaucoma.Ageing Res Rev. 2016; 29: 26-41 PubMed Abstract \| Publisher Full Text References used by the Authors in this Reply 1. van Schilfgaarde M, van Alphen L, Eijk P, Everts V, Dankert J. Paracytosis of Haemophilus influenzae through cell layers of NCI-H292 lung epithelial cells. Infect Immun. 1995 Dec;63(12):4729–4737. PMCID: PMC173678 2. Johnson M, Erickson K. Mechanisms and routes of aqueous humor drainage. Principles and Practice of Ophthalmology. Saunders Philadelphia; 2000;4:2577–2595. 3. Braakman ST, Pedrigi RM, Read AT, Smith JAE, Stamer WD, Ethier CR, Overby DR. Biomechanical strain as a trigger for pore formation in Schlemm’s canal endothelial cells. Exp Eye Res. 2014 Oct;127:224–235. PMCID: PMC4175173 4. Saccà SC, Gandolfi S, Bagnis A, Manni G, Damonte G, Traverso CE, Izzotti A. The Outflow Pathway: A Tissue With Morphological and Functional Unity. J Cell Physiol. 2016 Sep;231(9):1876–1893. PMID: 26754581 5. Ethier CR, Coloma FM, Sit AJ, Johnson M. Two pore types in the inner-wall endothelium of Schlemm’s canal. Invest Ophthalmol Vis Sci. 1998;39:2041–2048. 6. Alvarado JA, Betanzos A, Franse-Carman L, Chen J, González-Mariscal L. Endothelia of Schlemm’s canal and trabecular meshwork: distinct molecular, functional, and anatomic features. Am J Physiol Cell Physiol. 2004 Mar;286(3):C621–34. PMID: 14613887 7. Johnstone MA, Grant WM. Pressure-dependent changes in structures of the aqueous outflow system of human and monkey eyes. Am J Ophthalmol. 1973 Mar;75(3):365–383. PMID: 4633234 8. Tamm ER. The trabecular meshwork outflow pathways: structural and functional aspects. Exp Eye Res. 2009 Apr;88(4):648–655. PMID: 19239914 9. Braakman ST, Daniel Stamer W, Overby DR. A fluorescent permeability assay for Schlemm’s canal endothelial cells in response to stretch. Invest Ophthalmol Vis Sci. The Association for Research in Vision and Ophthalmology; 2014 Apr 30;55(13):5983–5983. 10. Llobet A, Gasull X, Gual A. Understanding trabecular meshwork physiology: a key to the control of intraocular pressure? News Physiol Sci. 2003 Oct;18:205–209. PMID: 14500801 11. Dang Y, Waxman S, Wang C, Loewen RT, Sun M, Loewen N. A porcine ex vivo model of pigmentary glaucoma. Sci Rep [Internet]. 2018 Mar 26;8. Available from: http://dx.doi.org/10.1038/s41598-018-23861-x 12. Pattabiraman PP, Rao PV. Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells. Am J Physiol Cell Physiol. 2010 Mar;298(3):C749–63. PMCID: PMC2838580 13. Tanihara H, Inoue T, Yamamoto T, Kuwayama Y, Abe H, Suganami H, Araie M, K-115 Clinical Study Group. Intra-ocular pressure-lowering effects of a Rho kinase inhibitor, ripasudil (K-115), over 24 hours in primary open-angle glaucoma and ocular hypertension: a randomized, open-label, crossover study. Acta Ophthalmol. 2015 Jun;93(4):e254–60. PMID: 25487877 14. Tanihara H, Inoue T, Yamamoto T, Kuwayama Y, Abe H, Araie M, K-115 Clinical Study Group. Phase 1 clinical trials of a selective Rho kinase inhibitor, K-115. JAMA Ophthalmol. 2013 Oct;131(10):1288–1295. PMID: 23787820 15. Epstein DL, Freddo TF, Anderson PJ, Patterson MM, Bassett-Chu S. Experimental obstruction to aqueous outflow by pigment particles in living monkeys. Invest Ophthalmol Vis Sci. 1986 Mar;27(3):387–395. PMID: 3949467 16. Campbell DG, Simmons RJ, Grant WM. Ghost cells as a cause of glaucoma. Am J Ophthalmol. 1976 Apr;81(4):441–450. PMID: 1266922 17. Moorthy RS, Mermoud A, Baerveldt G, Minckler DS, Lee PP, Rao NA. Glaucoma associated with uveitis. Surv Ophthalmol. 1997 Mar;41(5):361–394. PMID: 9163835 18. Callender D, Jay JL, Barrie T. Schwartz–Matsuo syndrome: atypical presentation as acute open angle glaucoma. Br J Ophthalmol. BMJ Publishing Group Ltd; 1997 Jul 1;81(7):608–608. 19. Lee RK. The molecular pathophysiology of pseudoexfoliation glaucoma. Curr Opin Ophthalmol. 2008 Mar;19(2):95–101. PMID: 18301281 20. Ritch R, Schlötzer-Schrehardt U, Konstas AGP. Why is glaucoma associated with exfoliation syndrome? Prog Retin Eye Res. 2003 May;22(3):253–275. PMID: 12852486 21. Gottanka J, Johnson DH, Grehn F, Lütjen-Drecoll E. Histologic findings in pigment dispersion syndrome and pigmentary glaucoma. J Glaucoma. 2006 Apr;15(2):142–151. PMID: 16633228 22. Alvarado JA, Murphy CG. Outflow obstruction in pigmentary and primary open angle glaucoma. Arch Ophthalmol. 1992 Dec;110(12):1769–1778. PMID: 1463421 23. Zhou L, Li Y, Beatrice Y J. 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Invest Ophthalmol Vis Sci. 1986 Mar;27(3):387–395. PMID: 3949467 27. Abu-Hassan DW, Acott TS, Kelley MJ. The Trabecular Meshwork: A Basic Review of Form and Function. J Ocul Biol Dis Infor [Internet]. 2014 May;2(1). Available from: https://www.ncbi.nlm.nih.gov/pubmed/25356439 PMCID: PMC4209746 28. Nakajima E, Nakajima T, Minagawa Y, Shearer TR, Azuma M. Contribution of ROCK in contraction of trabecular meshwork: proposed mechanism for regulating aqueous outflow in monkey and human eyes. J Pharm Sci. 2005 Apr;94(4):701–708. PMID: 15682386 29. Wang J, Liu X, Zhong Y. Rho/Rho-associated kinase pathway in glaucoma (Review). Int J Oncol. 2013 Nov;43(5):1357–1367. PMID: 24042317 30. Doornaert B, Leblond V, Planus E, Galiacy S, Laurent VM, Gras G, Isabey D, Lafuma C. Time course of actin cytoskeleton stiffness and matrix adhesion molecules in human bronchial epithelial cell cultures. Exp Cell Res. 2003 Jul 15;287(2):199–208. PMID: 12837276 31. Wang K, Read AT, Sulchek T, Ethier CR. Trabecular meshwork stiffness in glaucoma. Exp Eye Res. 2017 May 1;158:3–12. 32. Saccà SC, Gandolfi S, Bagnis A, Manni G, Damonte G, Traverso CE, Izzotti A. From DNA damage to functional changes of the trabecular meshwork in aging and glaucoma. Ageing Res Rev. 2016 Aug;29:26–41. PMID: 27242026 33. Liton PB, Lin Y, Gonzalez P, Epstein DL. Potential role of lysosomal dysfunction in the pathogenesis of primary open angle glaucoma. Autophagy. 2009 Jan;5(1):122–124. PMCID: PMC2745819" } ] } ]	1	https://f1000research.com/articles/7-174
https://f1000research.com/articles/7-431/v1	06 Apr 18	{ "type": "Software Tool Article", "title": "Authoring Bioconductor workflows with BiocWorkflowTools", "authors": [ "Mike L. Smith", "Andrzej K. Oleś", "Wolfgang Huber", "Andrzej K. Oleś", "Wolfgang Huber" ], "abstract": "The Bioconductor Gateway on the F1000Research platform is a channel for peer-reviewed and citable publication of end-to-end data analysis workflows rooted in the Bioconductor ecosystem. In addition to the largely static journal publication, it is hoped that authors will also deposit their workflows as executable documents on Bioconductor, where the benefits of regular code testing and easy updating can be realized. Ideally these two endpoints would be met from a single source document. However, so far this has not been easy, due to lack of a technical solution that meets both the requirements of the F1000Research article submission format and the executable documents on Bioconductor.\nSubmission to the platform requires a LaTeX file, which many authors traditionally have produced by writing an Rnw document for Sweave or knitr. On the other hand, to produce the HTML rendering of the document hosted by Bioconductor, the most straightforward starting point is the R Markdown format. Tools such as pandoc enable conversion between many formats, but typically a high degree of manual intervention used to be required to satisfactorily handle aspects such as floating figures, cross-references, literature references, and author affiliations. The BiocWorkflowTools package aims to solve this problem by enabling authors to work with R Markdown right up until the moment they wish to submit to the platform.", "keywords": [ "Bioconductor", "workflow", "reproducible research", "markdown", "scientific publishing" ], "content": "Introduction\n\nBioconductor workflow vignettes are educational resources that demonstrate how one might tackle a particular multi-step bioinformatic analysis, primarily (but not necessarily exclusively) using the software found in the Bioconductor project1. They expand on the vignettes found in individual software packages by focusing on how multiple tools can be combined to conduct an analysis from beginning to end, rather than highlighting the features of a single resource. However they do share many similarities, in particular the desire to write such workflows in a literate programming style, with explanatory text surrounding executable code. This provides benefit to the reader, who can see each step of a workflow in context, and to the author, who can periodically check that the code is still valid and make changes to reflect either updates to the software they rely on, or improvements in methodology. These documents are then hosted on the [Bioconductor website] (www.bioconductor.org), which provides a centralized location for readers to find the articles and to download the software packages detailed within them. Workflow authors are encouraged to also submit their work as an article to F1000Research’s Bioconductor Gateway, which provides the benefits (both to authors and readers) of increased visibility, peer-review and a citable reference. The intention is that (essentially) identical content will be present in both locations.\n\nHowever, the requirements of the two publishing platforms are distinct. In order to regularly check code functionality and provide a workflow that is straight-forward to download and run by users, Bioconductor needs to be provided with documents written in R Markdown2 or Sweave3, which are compatible with the standard literate programming engines available for R. On the other hand, F1000Research request submissions in LATEX or Microsoft Word format, where the code cannot be run directly. Both parties also apply their own style and branding to the final documents to present a coherent portfolio to end-users.\n\nGiven these distinct requirements, it has been somewhat difficult for an author to maintain a single document for submission to both platforms. This commonly results in prioritization of one over the other, followed by a non-trivial effort to convert to the other. Alternatively the author faces the challenge of writing two documents at the same time, trying to keep the information content synchronized, whilst dealing with two rather different syntaxes for document layout and formatting.\n\nHere we present a strategy and accompanying tools to help authors develop and maintain a single document that can easily be transformed into the required format for submission to either platform.\n\n\nMethods\n\nGiven the intention for workflow documents to be full of executable examples that can be regularly checked and updated as necessary, it seems natural to recommend working with one of the literate programming formats available in R, rather than using a static typesetting tool. As previously mentioned, there are two formats commonly used here: Sweave and R Markdown. This immediately presents an author with a choice, even before a single word has been written, and there are reasonable arguments for electing to choose either; R Markdown has a simpler syntax and can be easily transformed into HTML for display on a website, while Sweave offers more precise control over document formatting and can readily be converted into a LATEX format suitable for journal submission.\n\nIn order to streamline this, we have chosen to support only R Markdown as an input format, since this can be directly submitted to Bioconductor, with the conversion into the HTML format displayed on the website handled on their side. This then leaves the challenge of converting R Markdown into a format suitable for journal submission. To tackle this we have developed BiocWorkflowTools, an R package that provides article templates, conversion tools and the ability to upload documents to Overleaf.com (F1000Research’s preferred LATEX submission system).\n\nIn order to use BiocWorkflowTools the user must already have R version 3.4.0 or newer installed on their system. We also recommend working in the RStudio environment, however this is optional and all operations can be carried out at the command line with instructions for both approaches provided below.\n\nBiocWorkflowTools can be obtained from the Bioconductor package repository by running the following commands in your R session.\n\n\n\nGiven BiocWorkflowTools’s raison d’être is to ease the burden of meeting the distinct requirements of two publishing platforms in a hassle-free manner as possible, our recommended strategy assumes that most authors begin a project with the intention of submitting the final outcome to both Bioconductor and F1000Research.\n\nFor F1000Research, the list of material required is straight-forward and familiar: the article itself, a list of references, figures, and supplementary materials. These can then be sent as a collection of files. When it comes to Bioconductor all the same materials are required; however their computing infrastructure, which enables the regular document checking and easy distribution, also requires that the submission is made in the form of an R package. There are numerous resources discussing how to create an R package4 (and we would highly recommend potential authors to read these if they are not familiar with writing packages), but to streamline this process we provide the function createBiocWorkflow, which will create the minimum folder structure needed for submission to Bioconductor.\n\n\n\nRunning the example above will create a workflow package called MyWorkflow with the subdirectory vignettes containing an article template named MyWorkflow.Rmd. It is in this file that one should start developing their workflow document. In its initial state the template provides an exemplary skeleton of a typical workflow article, along with examples of how to include specific document features such as figures, tables, formulae and code blocks, in much the same way as the more traditional LATEX and Microsoft Word templates available from F1000Research’s website. The template also includes an example of the required document header, where article metadata including the title, author names, their affiliations and the abstract are specified.\n\nIn the example above, changing the argument open = TRUE will open a new RStudio project rooted in the newly created MyWorkflow folder.\n\nIf you do not wish to make a complete package, and instead would simply rather use R Markdown to author an F1000Research article, the recommended platform for most authors is still to work in RStudio. Rather than creating a new package as before, the user can opt to create a new R Markdown document from the file menu and, assuming the BiocWorkflowTools package has been installed, will be presented with the option to use the F1000Research Article template (Figure 1).\n\nThe F1000Research template can be accessed via the ‘New R Markdown’ file menu dialog.\n\nThis will automatically open a new document based the F1000Research template described previously.\n\nWorking outside RStudio Even if you choose not to work in the RStudio environment, you can still use the included template to create a new file. We recommend using the template as a starting point to facilitate adherence to the required article structure. The command below will create a folder named MyArticle within the current working directory, and this in turn will contain the template MyArticle.Rmd which one can edit with the tool of choice.\n\n\n\nIf you are using RStudio to edit your workflow, the simplest way to create the LATEX version of your document is to press the ‘Knit’ button above the document pane in your workspace. This will process the document and generate both the LATEX version and a compiled PDF so you can see how the final print version will look. This process also carries out some necessary housekeeping, such as copying the required F1000Research LATEX style file and the journal logo into the same location as your document, so you may notice some additional files appearing.\n\nIf one prefers to work in an editor other than RStudio, it is still possible to transform the R Markdown file into LATEX (along with the aforementioned housekeeping) by using the function render from the rmarkdown package. This will carry out much the same conversion process as using RStudio, executing the code chunks and producing the expected LATEX and PDF output files.\n\n\n\nIdeally submission of a workflow is to both Bioconductor and F1000Research. Instructions for contributing to Bioconductor are available from https://www.bioconductor.org/developers/how-to/workflows.\n\nSubmission of LATEX articles to F1000Research is currently performed using Overleaf.com, an online collaborative writing and publishing tool. Complete details are available online, but assuming one has already written an R Markdown workflow and generated a LATEX source file, they can choose to upload the file directly to Overleaf using their web browser.\n\nBiocWorkflowTools provides the function uploadToOverleaf as an alternative option for getting the article into the Overleaf system. This function takes the document directory and sends this to Overleaf, creating a new project for you automatically. The function will directly open the new project in a web browser.\n\n\n\nBiocWorkflowTools::uploadToOverleaf(\"MyArticle\")\n\nAt this point it is important to point out that both the LATEX and R Markdown versions of the article are present in the Overleaf project, with the first of these being rendered into the document preview one sees on the site. In the Overleaf environment, only changes to the LATEX version will be reflected in the preview pane, rather than the R Markdown the author has been working with until now. Thus it is easy for the two documents to become out of sync if edits are made using the browser interface. For this reason we recommend only working with Overleaf for the final submission process, and eschewing its document editing features at this point. Overleaf offers many attractive features such as collaborative editing between authors (particularly those who may not be familiar with the Rstudio environment) and live rendering of changes. However it should be emphasised again that it is natural to want to edit the LATEX document in the Overleaf environment, which undermines the primary motivation behind BiocWorkflowTools, and as such our recommendation is to work exclusively on the Rmarkdown version of the article (using whichever version control system and collaborative tools the authors prefer) and then use Overleaf purely as a submission tool.\n\nAssuming the article is provisionally accepted, the journal editors will create a second, private, Overleaf repository for minor copy editing. Editorial comments and instructions will be included in the R Markdown document, enclosed in comment tags e.g. <!-- Editorial comment -->.\n\nAt this stage one can make changes to the R Markdown document in the web browser interface, however there is currently no way to regenerate the LATEX containg the changes from here. Instead, to make additional changes after an Overleaf project has been created, we recommend utilising the fact that all Overleaf projects can be interfaced using git. Instructions for initialising this on your local machine provided by Overleaf5. Since this is a private project the author will need to supply their Overleaf username and password to use git. If the manuscript is already under git version control, the Overleaf server can be added as an additional remote repository. Authors can then work on the manuscript offline, and use the regular git commands to keep the local copy in sync, such as git pull to get the latest version from Overleaf. Before committing any edits made to the source Rmd file remember to also regenerate the LATEX output file. Once they are ready to push the changes back to Overleaf, they will instantly appear online.\n\n\n\nOnce the author is happy they have addressed the editor’s comments resubmission can be performed via Overleaf.\n\n\nSummary\n\nBiocWorkflowTools provides a straightforward set of helper functions and a document template discoverable in RStudio intended to simplify the process of authoring a source document that can both be run as an executable document, and also submitted to a journal for publication. We have focused on the collaboration between Bioconductor and F1000Research, and the authoring of R Markdown workflows. By supporting R Markdown and integrating the document template with RStudio, we hope we have made the path to meeting the requirements of both publication formats simpler for authors. Currently two distinct documents are still required for submission (R Markdown file, and the LATEX file derived from this), but the potential exists to move the use of BiocWorkflowTools from the author to the journal, thus removing this final hurdle to the ambition of submitting a single source to both locations.\n\nAlthough BiocWorkflowTools currently only supports the F1000Research article format, support for other scientific journals can be broadened in the future with the addition of more templates to the package, without requiring changes to the underlying workflow. The authors welcome approaches from publications that are interested in supporting the submission of R Markdown documents.\n\nTo demonstrate and test the tool’s utility, this article has been written entirely in R Markdown.\n\n\nSoftware availability\n\nSoftware available from: http://bioconductor.org/packages/BiocWorkflowTools\n\nSource code available from: https://github.com/grimbough/BiocWorkflowTools\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.12086076\n\nSoftware license: MIT", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nMLS is funded by The German Network for Bioinformatics Infrastructure (de.NBI) Förderkennzeichen Nr. 031A537 A. AKO is funded by the Federal Ministry of Education and Research (BMBF) grant no. 01EK1502A (BioToP) and the European Union Horizon 2020 research and innovation program under grant agreement no. 633974 (SOUND project).\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 Michael I. Love for sharing his experience with submitting workflows to both locations and his approaches to document format conversion. They would also like to thank the F1000Research editorial team for their collaboration in determining how to work with R Markdown as the baseline article format.\n\n\nReferences\n\nHuber W, Carey VJ, Gentleman R, et al.: rchestrating high-throughput genomic analysis with Bioconductor. Nat Methods. 2015; 12(2): 115–121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllaire JJ, Xie Y, McPherson J, et al.: rmarkdown: Dynamic Documents for R. R package version 1.7. 2017. Reference Source\n\nLeisch F: Sweave. dynamic generation of statistical reports using literate data analysis. 2002. Reference Source\n\nWickham H: R packages: organize, test, document, and share your code. \"O’Reilly Media, Inc.\", 2015. Reference Source\n\nOverleaf.com. Date Accessed: 21-03-2018. Reference Source\n\nSmith ML, Oleś AK: grimbough/BiocWorkflowTools: F1000 Article Submission (Version F1000). Zenodo. 2018. Data Source" }	[ { "id": "32898", "date": "26 Apr 2018", "name": "Justin Kitzes", "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 new package, BiocWorkflowTools, that is designed to allow authors to write a manuscript in a single R Markdown file and then easily submit the manuscript to both F1000Research and Bioconductor, two platforms with different submission requirements. The goal is to reduce the risk that versions of the manuscript published in both places will begin to inadvertently diverge. The package addresses an important and somewhat general problem in scientific publishing, which is how to keep multiple versions of a published article in sync through the revision and publication process.\n\nThe article is well-written and describes the use of BiocWorkflowTools clearly. I have several major and minor comments on the article. These mainly request clarification or expansion of the points made in the manuscript, and none should stand in the way of the article’s acceptance.\n\nMajor comments\n\nAs the article is brief, it does not provide instructions for the many associated steps that could be necessary to use the package, such as how to structure an R package, set up an Overleaf account, write in Markdown, write in LaTeX, and use git (both locally and remotely). It would seem reasonable, though, to assume that the target audience for the package will already have these skills.\n\nWhile I understand the problem that the package is attempting to solve, I might also question the wisdom of a publication strategy that requires this package at all. That is, I question whether it’s a good idea, even with this package, to attempt to duplicate nearly identical content across two platforms. I can see the value added by having an article published on the F1000 platform, but is the value of being able to execute the embedded code blocks really worth the trouble of duplicating the entire article on Bioconductor?\nAs an alternative, although it would take more effort, what about having Bioconductor scrape the HTML from F1000 (or finding a way to access the article text in a structured format) and extract code blocks when the manuscript occasionally needs to be executed? This would add burden on the Bioconductor side but would allow authors to only worry about a single public document.\n\nI’m curious as to why the package developers chose to go through LaTeX and Overleaf for F1000 submission rather than the Word route (e.g., by converting R Markdown to Word and then having authors submit the Word version to F1000). I would be slightly concerned that involving another third party (Overleaf) increases the potential for the package breaking in the future due to changes to the Overleaf API, Overleaf’s default package list, the LaTeX packages themselves, F1000’s relationship with Overleaf, etc. In comparison, the DOC/DOCX format is relatively stable. I was more enthusiastic about the Overleaf workflow until reading all of the caveats and warnings surrounding the appropriate approach to making revisions in Overleaf, and how that can cause the very problems the package is designed to prevent.\n\nWould it be possible to add a function to the package that would automatically check for synchronization between the F1000 and Bioconductor versions of an article (this would likely involve web scraping as mentioned above)? If the two versions did go out of sync, it will likely be very difficult to detect that divergence.\n\nDoes the package correctly handle all of the issues described in the abstract in the conversion from Markdown to LaTeX, including “floating figures, cross-references, literature references, and author affiliations”?\n\nHow does this package work under the hood? In particular, is pandoc a dependency and can this package be understood as a kind of pandoc wrapper that handles the special cases listed above?\n\nMinor comments\n\nThere is an incorrectly written Markdown link that is currently appearing as “[Bioconductor website] (www.bioconductor.org)” - I believe the leading “http://” will be needed for this to render properly as a link.\n\nAwkward wording - “Ideally submission of a workflow is to both Bioconductor and F1000Research” could be “Ideally a workflow is submitted to both…”\n\nThere appears to be a duplicated line that appears both inside and outside of a code block: “BiocWorkflowTools::uploadToOverleaf(\"MyArticle\")”\n\nDo I understand correctly that the F1000 editors will make comments in an R markdown file that appears on Overleaf, but then expect revisions to occur in LaTeX? This seems to be an unusual setup, just confirming that it’s correct.\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": "34126", "date": "11 Jun 2018", "name": "Laurent Gatto", "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 BiocWorkflowTools Bioconductor package, a tool to write and maintain a single entry point that can be converted and submitted as a Bioconductor workflow or as a F1000Research Bioconductor gateway article.\nMy understanding is that the authors effectively want to address or facilitate three challenges with their packages:\n1) The technical challenge that is allowing authors to use R markdown to generate outputs that will fit the requirements set by Bioconductor (for the submission of an R package containing a workflow vignette) and F1000Research (submission of a LaTeX document). This purely technical goal is elegantly achieved thanks to the BiocWorkflowTools package that leverages existing infrastructure and templates.\n2) The authors also seem to favour submission of workflows to both Bioconductor and F1000Research, so that authors and readers will benefit from the advantages of both platforms, i.e. reproducibility from the former, and visibility and citable reference from the latter.\nHowever, here I feel that the solution falls short because these two options rely on two pipelines in BiocWorkflowTools, namely\nBiocWorkflowTools::createBiocWorkflow for Bioconductor workflows and rmarkdown::draft(template = \"f1000_article\") for an F1000Research article.\nThe problem I see is that this will essentially define from the beginning what distribution platform future workflow authors will favour, instead of being able to write the workflow with BiocWorkflowTools and then submit to both locations.\nTechnically, the article corresponds to the vignette of the R package. The package also provides additional meta-data in the DESCRIPTION file. It would be easy for a user that has experience in package development to convert one in the other, but I am wondering if a single pipeline wouldn't be a better option for authors that don't have any experience in package development. Specifically, I am thinking of either converting the Rmd article to a package (by populating the DESCRIPTION file using meta-data in the article's header), or choosing the package as single pipeline and uploading the vignette directory using BiocWorkflowTools::uploadToOverleaf.\n3) The last issue, arguably the most difficult one to address, is to bridge the gap between the fully reproducible scientific document and the manual publishing pipeline.\nIronically, the greatest breakthrough here seems to be that \"Editorial comments and instructions will be included in the R Markdown document\" using Overleaf as an intermediate broker. To avoid breaking the reproducible pipeline, authors are advised to use the Overleaf project (which is also a git repository) as a remote and pull changes into their local repository.\nEstablishing a pipeline that maintains reproducibility and traceability beyond submission is very important. While it isn't strictly part of the tool, it would be valuable to describe how to set this up in a bit more details, or provide links to useful resources.\nAnother dimension that could be documented is when workflow authors already work collaboratively on the article/package using a git repository via, for example, GitHub. They would have their local/remote repositories for the duration of the writing and, when ready to submit, upload to an Overleaf project (that itself is a remote git repository). Editorial comments are then returned in a new Overleaf project/github remote repository. Authors would then add one (or two) additional remote repositories pointing to the Overleaf project(s). Finally, it would be useful to also describe how a revision of the article would be updated and resubmitted via Overleaf.\nMy suggestions for this manuscript are two-fold:\n1. Comment on the usefulness of having two independent workflow pipelines (one through a package for Bioconductor, and another one through an Rmd file for F1000Research) or a single one. I fear that having two will limit the submission to both locations.\n2. Even though it isn't directly related to the BiocWorkflowTools package, it would be very useful for the authors to provide more details and/or links to relevant resources to further integrate the local workflow repository to the remote Overleaf project to maintain reproducibility beyond the first submission.\nMinor comments:\n- The first letter of 'Orchestrating' is missing in the title of Ref. 1. - The link to Bioconductor in the first paragraph is mis-formatted. - The code in the code chunk describing how to upload the article to Overleaf is repeated.\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/7-431
https://f1000research.com/articles/7-428/v1	06 Apr 18	{ "type": "Research Article", "title": "Based on the perceptions of community stakeholders, how can adolescent pregnancies be prevented? A qualitative study", "authors": [ "Sadudee Phuhongsai", "Somdej Pinitsoontorn", "Sadudee Phuhongsai" ], "abstract": "Background: Adolescent pregnancy an important problem in adolescent health and government agencies need to focus on solving problem. The purpose of this research was to survey the perceptions of community stakeholders concerning the prevention of adolescent pregnancies in rural communities. Methods: Data collection was performed using group meetings with 103 stakeholders involved in adolescent pregnancy prevention. They were nurses, public health officials, parents or guardians, students, teachers, public health volunteers and community leaders. Thematic analysis indicated work on adolescent pregnancy prevention problems in rural areas was carried out by only some agencies such as district and sub-district health promoting hospitals, providing youth-friendly health service clinics and educating student leaders in schools on sex education. Results: Collectively, these results draw attention to the need for an appropriate program to strengthen adolescent, family and practitioner skills for the prevention of teenage pregnancies. Schools provide sex education as part of a health education curriculum, and some schools provide additional instruction in guidance classes. Problems from inconsistent work when networks fail were encountered. Stakeholders believe adolescent pregnancy prevention should focus on the following: (1) adolescents should receive training for skill development with content related to knowledge about sex, negotiation, refusal, morality and ethics, (2) teachers should receive training on comprehensive sexual education, and, (3) families should work to improve their communication on sexual health and development Conclusions: There is a strong need for families to develop the ability to communicate with each other about sexuality and reproductive health. Developing parenting skills on how and when to talk about sex with their adolescents and open parental communication on sexuality issues at home is necessary. Activities need to also be developed for adolescents who are more inclined to engage in risky sexual behaviors.", "keywords": [ "Adolescent", "Pregnancy", "Stakeholder", "Community Participation", "Planning" ], "content": "Introduction\n\nApproximately, two million adolescent females aged under 15 years become pregnant in developing regions every year, according to World Health Organization (WHO) figures from 2018. Almost four million adolescent females in this age group undergo unsafe abortions with complications (See WHO fact sheet on Adolescent pregnancy). According to data from the WHO, the adolescent birth rates in the various countries worldwide ranges from 1–200 per thousand girls. Adolescent pregnancy remains a major cause of death among mothers and children while contributing to the cycle of ill-health and poverty (WHO, 2018). In the three years from 2012 – 2014, Thailand had adolescent pregnancy rates of 53.4, 51.2 and 47.9 per thousand among adolescent women aged 15–19 years, respectively (Tantisewas et al., 2014).\n\nRegarding guidelines for solving the aforementioned problem, the WHO and the United Nations Population Fund (UNFPA) published guidelines in 2011. They recommended that political leaders, planners and community leaders build understanding and support for reducing pregnancies among women under 20 years of age (United Nations Population Fund (UNFPA) & Roger I, 2016; WHO, 2014). Thailand recognized the seriousness of its adolescent pregnancy problems. Its government set policies to address this problem, in addition to promoting social measures that attempt to curb adolescent pregnancies. These include more comprehensive sex education, allowing students who have become pregnant to finish their studies, control of the dissemination of pornography, and greater parental involvement in their children’s lives. Consequently, each ministry is required to have working guidelines to reduce the incidence of teenage pregnancies. (Tantisewas et al., 2014).\n\nBased on performance evaluation, no clear and integrative problem management approach has been found effective. Policies are inconsistent and efforts are both wasteful and complicated (Chamrunsawat, 2014; UNICEF, 2016). Adolescents have little participation in these programs. Each ministry functions slightly differently and there is little communication between them (Health Assembly, 2016; Poonkhum et al., 2010). Comprehensive sex education and adolescent-friendly activities are not being conducted in every educational facility. Activities in the areas of physical and social relations are not being presented to adolescents at risk. Furthermore, there is a general lack of involvement among the relevant government agencies (Chamrunsawat, 2014) in improving adolescent health. Addressing these issues must begin from the early adolescent period (ages 10–14) (Chandra-Mouli et al., 2015). Many factors are related about adolescent pregnancy preventions. For example, at a personal level, knowledge, skills and empowerment must be developed. Furthermore, safe places must be created where young people may go and be free of sexual pressures imposed by their peers. Family and friends need closer relationships and a high degree of communication. Communities and local organizations need to provide convenient services and opportunities for adolescents in places such as schools (Svanemyr et al., 2015; WHO, 2014). Additionally, youth-friendly guidelines need to be developed based on community needs (Cassell et al., 2005).\n\nThere are teenagers who do not see school as important. Among some adolescent females, there is a positive attitude about becoming pregnant, even outside of marriage. If a girl becomes pregnant, it is unlikely that she will continue with her studies. Focus must be placed on changing negative social values and norms along with developing adolescent sexual and reproductive health. This can be done by raising awareness, acceptance and support for youth-friendly sexual and reproductive health education services. Gender inequality, in terms of beliefs, attitudes and norms, needs to be addressed (Chandra-Mouli et al., 2015) in addition to creating connections that link services in various settings, such as schools, to promote utilization of these services (Denno et al., 2015). At the broadest societal level, efforts to concurrently build awareness of adolescent sexual and reproductive health must be made through mass media approaches.\n\nTherefore, efforts to improve adolescent health must be made by recognizing and using a variety of driving mechanisms (Kuruvilla et al., 2016). From the aforementioned principles, participation is an excellent method for planning projects and policies because it helps ensure the strategies developed are appropriate for the targeted age group in culturally and geographically diverse communities. Support for stakeholder participation in the target groups creates more effective results and combines democratic principles in the decision-making process as well as maintaining strong partnerships (Tevendale et al., 2017). For example, a study of community participation in a campaign for prevention of teenage pregnancies found that parents in communities changed their attitudes about sex, had more positive thoughts, communicated with and understood adolescents to a greater degree than before the project (Phoochaemchot & Chomnirat, 2012). Additionally, a study that implemented community-based empowerment interventions found that the project could be improved with earlier active participation by unmarried pregnant adolescents and increased support by parents (Leerlooijer et al., 2013).\n\nAlthough we have a good understanding of adolescent needs and problems, there are still many gaps in our knowledge and understanding (UNICEF, 2016). Based on evaluations and interventions related to prevention of adolescent pregnancies, evidence-based practices remain important for outlining a national policy framework (Lavin & Cox, 2012). Thus, the purpose of this research was to study stakeholders’ perceptions about prevention of adolescent pregnancies and planning for teenage pregnancy prevention with community participation.\n\n\nMethods\n\nThis study on the perceptions of participants included public health officers, nurses from heath promoting sub-district hospitals, district hospitals, public health officers, public health volunteers, key persons in the communities, teachers, representative students and parents. There were 103 persons purposively selected. The participants were involved in adolescent pregnancy problems or part of adolescent pregnancy reduction operations from hospitals, schools and communities. The researcher had issued self-introduction letter from Khon Kaen University in order to meet with Director of Public Health Officer, Director of hospitals and Director of the secondary schools of each province and to explain purposes of the research. They also included; for hospitals, invitations for personnel relating to teenage pregnancy reduction operations in hospitals and the community, for schools, invitations to teachers, students and parents or guardians involved in teenage pregnancy reduction operations to participate in the conference and group discussion. People from these communities all volunteered to participate in the study\n\nThe communities under study were Kalasin, Khon Kaen, Mahasarakham and Roi-et provinces. The adolescent birthrate of these areas during 2011–2013 was 35–54 per 1,000 women aged 15–19 years old. (Bureau of Reproductive Health, 2014) All 4 provinces are located within central area of Thailand´s northeastern region. Volunteered participants taking parts in the research in Kalasin were from district region and sub-district region for Khon Kaen, Mahasarakham, and Roi Et. Data used in the research were collected from September-November 2014.\n\nThe research instruments consisted of conferences, documents, field notes and stakeholder responses captured using audio recorders. Documents used were summary results of the survey on sexual behaviors in students Grade 7 and 8, which was collected 3 months prior to the group discussion, and reports on operation results of youth friendly health service clinic from district hospital in Kalasin province.\n\nThis initial study was conducted in June-July 2014 in district of Kalasin and sub-district of Khon Kaen, Mahasarakham, and Roi Et Province, central northeastern Thailand. Participants were students in grade 7 and 8. 624 students were included in the study. The study was explained to the students and parental consent forms were given. After both students and their parents give written informed consent the student were given a self-administered questionnaire to fill out. Exclusion criterion was failure to give consent. The study was approved by the Ethics Committee of Khon Kaen: code 571119. The questionnaire asked about demographics characteristics and sexual risk behavior (Questionnaire is available as Supplementary File 1). Students who had sexual intercourse were asked about their first sex age, contraception, STI, pregnancy and abortion. Questionnaire data were entered into Microsoft Excel 2008 in duplicate. The data were frequencies and percentage.\n\nData were collected from group discussions (conducted by a public health officer) using participatory activities on four occasions in the four communities mentioned above. The number of participants in each group was 25–28 persons. Each session was conducted for 6–7 hours in which the participants shared their opinions about prevention of adolescent pregnancies. The participatory activities consisted of introduction, adolescent pregnancy problem analysis, plan of problem solving and presentation of plans.\n\nData was collected as part of empowerment evaluation of the program to reduce teenage pregnancy in central northeastern Thailand. Instruments used in data collection were adapted from Empowerment Evaluation Principles in Practice. The researcher utilized and adapted the concept of Empowerment Evaluation from David M. Fetterman (Fetterman DM, 2001, Fetterman DM, Abraham Wandersman A, 2005) to determine activities and activity planning conferences for teenage pregnancy reduction project. Activities and contents were adapted to suit issues within the study areas, with activity details as follows:\n\n1. Mission review process included identifying/determining things that needed to be done together and the common mission.\n\n2. Activity review and prioritizing process was to analyze weaknesses and strengths of the project (Taking Stock)\n\n3. Planning for the future.\n\nFor Kalasin, the conference location was the district hospital´s conference hall. For Khon Kaen, Mahasarakham, and Roi Et, the conferences were held at the sub-district secondary school conference rooms. The conference took 6–7 hours, with a team of 1 lecturer and 4 facilitators consisting of 1 university professors with expertise in evaluation, a public health officer with over 10 years of experiences as a facilitator who conducted the group discussion, 1 professional nurse, and 2 doctoral students (1 doctoral student was a researcher SP). Activities during the conference consisted of 4 stages as follows:\n\nPart 1: Introduction; The activity began with introduction of each researcher, research purposes, lecturer team members, participants, and survey results regarding sexual behaviors in secondary schools within each province´s area for the participants to acknowledge the current situation.\n\nPart 2: Adolescent pregnancy problem analysis was conducted by each participant voicing their opinions regarding pregnancy issues within the region, causes, circumstances (as established by survey results) or emotions towards the issue. The second activity was stopped when no more new opinions were being provided. The duration was approximately 1 hour.\n\nPart 3: Plan of problem solving was an activity where the participants determined necessary things that needed to be done together and prioritized activities. The participants were divided into 3 subgroups: One group consisted of persons relating to public health and hospital operation. One group consisted of persons relating to school operation. One group consisted of persons relating to community operation. Activity duration was approximately 4 hours.\n\nPart 4: Presentation of plans was the presentation of the result gained from subgroup meeting and then gathered similar activities into one single project, and determined the person in-charge, which later on will be made into the adolescent pregnancy reduction plan for each area.\n\nGroup discussion were recorded and transcribed verbatim. Data was read and re-read. Theme form data were initially identify by the first author, and subsequently verified by both authors for coding consistency, emergence of main themes, and extraction of statements to support the themes.\n\nAll of the participants voluntarily agreed to share their opinions and had the right to ask questions before giving their written informed consent. The subjects were assured that their information would be kept confidential during data collection and analysis. This study was approved by the Khon Kaen University Ethics Committee in Human Research (HE 571119).\n\nAfter group discussion, the researcher sent the data summary back to all participants to check for data accuracy. The experienced researcher and agenda were the same in all four sessions. For audio recording, the tape was destroyed by the researcher after completion of the transcription and the data were summarized with no personal reference.\n\n\nResults\n\nThis study obtained results in the following three areas: (1) adolescent pregnancy prevention programs in hospitals and schools, (2) attitudes and perceptions of the adolescent pregnancy problem, and, (3) adolescent pregnancy prevention guidelines in the community.\n\nThe work to reduce pregnancies in communities is carried out by two agencies: (1) the district and sub-district health promoting hospitals and (2) the district and sub-district secondary schools.\n\nDistrict hospitals have the role of providing in-clinic teen services with youth-friendly health activities. These include counseling on reproductive health, promotion of condom use, sexual behavior risk assessment, pre-post HIV blood test counseling, nutrition, stress and general issues. Furthermore, services are provided in schools in the form of training sessions for student leaders to give them knowledge about helping adolescents delay sexual intercourse, sexual health education and community service programs to help parents and children have open communication about sexual health.\n\nThe sub-district health promoting hospitals provide consultation services on issues related to adolescents, distribution of condoms and referral services for emergency contraception, antenatal services and pregnancy termination in the cases where this is an option.\n\nDistrict and sub-district level secondary schools offer pregnancy prevention activities consisting of reproductive health education for one hour per week over four months, i.e., 16 unique lessons. Furthermore, student leaders received training on helping their peers delay sexual relations by providing instructions about refusal skills, negotiation and instructions on condom use, among other skills. Special activities are sometimes held on Valentine’s Day, since some students are more likely to have sex on this day.\n\nAll stakeholders recognized adolescent pregnancy as a significant problem. They consider adolescents difficult to understand and do not know how to communicate with them. This is evident from the following reports from parents:\n\nThe problem is caused by adolescents. Adolescents do not listen to what parents tell them. They only trust their friends.\n\n“My child is young. They doesn’t listen to anything I say. I (the parent) think premature pregnancies aren’t good but I haven’t dared to forbid him/her. I was afraid they will be upset with me. By the time I knew there was a problem, they was already 4–5 months pregnant. This makes it difficult to solve the problem.”\n\nFurthermore, according to the study, 30% of students in the community are being reared by their grandparents because their parents work in other provinces. Additionally, teenagers copied the risky sexual behaviors from peers. They also had access to pornography. From stakeholders’ opinion, these were important causes of early sexual encounters, leading to unplanned pregnancies.\n\nAdolescents also did not dare to communicate with parents. Students reported the following:\n\n“We don’t usually dare to speak to our parents about menstruation or pubic hair. I mostly studied independently from school and the Internet. But, I probably wouldn’t dare to go and ask my parents directly.”\n\n“I wanted to ask about lumps in my breasts and why they were there, but I was embarrassed. But, I don’t know what I’d ask for.”\n\nKey persons in communities had the opinion that people should observe adolescent behaviors, watching to see who adolescents relate with, especially friends from the opposite gender. The idea is that “it takes a village to raise a child, so the community will advise parents when their children engage in risky sexual behaviors”. However, these same key persons saw that community members took no role in the aforementioned activity.\n\nWith regard to views about prevention of adolescent pregnancies, stakeholders recognized the goal of ensuring that adolescents have good physical and psychological health, life skills, understanding of sexual health, healthy behaviors, and the support of parents. They agreed that communities should develop networks fostering adolescent sexual health.\n\nProblem-solving can be carried out by three groups, adolescents, families and networks.\n\nAdolescents – Activities to foster good morality and ethics in adolescents can be organized for adolescents to gain knowledge and understanding, form healthy attitudes and values about sex and avoid undesirable pregnancies through birth control measures. Adolescents should receive comprehensive sex education in school, and have increased access to condoms through mechanisms such as condom vending machines and public health volunteers. Furthermore, adolescents should engage in positive youth activities at schools. Annual sporting events should be organized in the community/between communities in the sub-district and at schools. These activities can be organized in combination with substance abuse education and mobile sex education for adolescents that are delivered outside of educational facilities.\n\nFamilies – Family-strengthening activities should emphasize communication between parents and adolescents. They can promote activities in which parents and adolescents jointly participate with the goal of strengthening community values.\n\nAdolescent Pregnancy Prevention Networks – Networks can be developed for strengthening community participation acting as special centers for addressing adolescents’ problems. They can also promote projects to develop adolescents’ understanding of the unplanned pregnancies. There should be participation from community agencies including school teachers/health instructors, public health officials, community leaders, student leaders and sub-district administrative organizations.\n\n\nDiscussion\n\nThis study is an expression of the stakeholder opinions regarding how pregnancy prevention should be carried out simultaneously among adolescents, families and networks.\n\nAccording to our findings, adolescents do not communicate with their parents. Knowledge, understanding, attitudes and value adjustment of stakeholders on sex and undesirable pregnancies should be created along with pregnancy prevention skill development. Teenagers should receive sex education in schools in addition to cultivation of morality and ethics consistent with principles of adolescent development. Furthermore, they should be supported to gain knowledge, skills and empowerment (Svanemyr et al., 2015; WHO, 2014).\n\nFamilies – The main problem is lack of communication on sexual topics within the family. According to studies in Thailand, parents are not likely to speak openly about sexual intercourse with their adolescents. (Nitirat, 2007; Sridawruang et al., 2010a) Studies also showed that parents experience difficulty in talking about sex and contraception with their children, or view sexual health as a topic that should not be discussed because of cultural traditions and beliefs (Sridawruang et al., 2010b) while other parents believed that their teens were still too young to learn and engage in sexual activity (Meechamnan et al., 2014). This study shows that adolescents also did not dare to communicate with parents. This idea is congruent to the study of Aroenthaweesub M. which found that the most difficult topics for adolescents in communicating with their parents were their personal issues such as opposite sex relationships and sexuality. (Aroenthaweesub & Hale, 2011) They feared the misunderstanding of the parents knowing that they were having sex and of being judged or rejected by their parents. (Meechamnan et al., 2014) Stakeholders had the opinion that emphasis should be placed on activities to strengthen families, enabling them to develop communications regarding sex, community values and morality. Families can participate in traditional activities in the community to build closer relationships (WHO, 2014).\n\nCommunity Work Problems – The findings indicated there is insufficient cooperation between government agencies, such as hospitals and schools with parents, guardians and community members. According to Chamroonsawadi K., the weaknesses of work to solve teen pregnancy problems are unclear policies, lack of clarification to create mutual understanding between government agencies, lack of integration of thought, inefficient use of budget from duplication of activities among the same target groups, lack of a meeting between the central administrators and local operators to create mutual understanding and sense of belonging in the same work (Chamrunsawat, 2014) UNFPA found that in Thailand the implementation of the problem of pregnancy in adolescents is a complex issue. Some agencies may not be familiar with the problem, and each agency had a different perspective on the nature of \"problems\" and \"solutions\" and maybe lack of sense \"ownership\". Work to solve teen pregnancy problems has found the problem of discontinuity in driving policy. Each agency does not see it as only their work and therefore not their direct duty to solve teen pregnancy problems often meaning they fail to continue to work on the issue. The plan to solve problems did not involve teenager or family participation (United Nations Population Fund (UNFPA) & Roger I, 2016; UNICEF, 2016). This is prudent as communities have an important role in improving adolescent health (Svanemyr et al., 2015; WHO, 2014). Additionally, friendly and guideline based communities should be formed (Cassell et al., 2005). Stakeholders thought that networks should be developed and strengthened, and that participation in the community should be through adolescent-friendly activities. Some examples include special centers for solving adolescent problems and projects to encourage adolescent pregnancy prevention with the participating community agencies engaging in self-assessment of their effectiveness. Some of the findings indicated that pregnancy prevention activities are comprised of promoting quality and access to youth-friendly reproductive health services, providing education on pregnancy prevention to stakeholders, and working with adolescents who engage in risky sexual behaviors. The community should analyze their problems and mobilize to develop solutions to their problems (Mueller et al., 2017). Additionally, other findings support the concept that community partners should support and organize community resources in the form of comprehensive, effective and sustainable programs (Cassell et al., 2005).\n\nHowever, the problem of access to pornography is difficult to control. Therefore, the government must work to find measures for controlling and preventing these media from being disseminated to adolescents. Youth can be educated so that they become aware of the dangers of pornography.\n\n\nConclusions\n\nAccording to stakeholders’ perceptions regarding adolescent pregnancies, the problems originate from adolescents, families, friends and the media. There are many organizations that work for prevention of adolescent pregnancies in rural communities. The results of this study suggest the need for participatory action by all agencies responsible for addressing the problem. There is a strong need for families to develop the ability to communicate with each other about sexuality and reproductive health such as develop the parenting skills on how and when to talk about sex with their adolescents, open parental communication on sexuality issue at home (communication skill of the parents) Activities need to be developed for adolescents who are more inclined to engage in risky sexual behaviors.\n\n\nData availability\n\nRaw datasets have not been made available at the request of the ethics committee in order to maintain participant confidentiality. Using Thai language as the national language of Thailand during the process of data gathering, all data including quotes are available in Thai translation and access to the complete raw data can be obtained upon request and with the permission of Ethics Committee of Khon Kaen University (www.ekku.ac.th). Anyone wishing to access the data should first contact the corresponding author who will facilitate contact with the ethical review board (Contact email:sampinit@hotmail.com)", "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\nStaff of the department of community medicine, faculty of medicine Khon Kaen University, health officers of the 4 province, and all participants that shared opinions and experiences to help the development of adolescent pregnancy preventions.\n\n\nSupplementary material\n\nSupplementary File 1 – Questionnaire used of in Grade 7 to 8 student survey.\n\nClick here to access the data.\n\n\nReferences\n\nAroenthaweesub M, Hale CL: Thai Family Communication Patterns: Parent-Adolescent Communication and the Well-Being of Thai Families. The First International Conference on Interdisciplinary Research and Development, 31 May – 1 June 2011, Thailand. Special Issue of the International Journal of the Computer, the Internet and Management. 2011; 19(SP1): 84.1–84.6. Reference Source\n\nBureau of Reproductive Health, Department of Health, Ministry of Public Health: Situation of reproductive health in adolescent. Reference Source\n\nCassell C, Santelli J, Gilbert BC, et al.: Mobilizing communities: an overview of the Community Coalition Partnership Programs for the Prevention of Teen Pregnancy. J Adolesc Health. 2005; 37(3 Suppl): S3–10. PubMed Abstract \| Publisher Full Text\n\nChamrunsawat K: Evaluation of the first national policy and strategy on reproductive health (2010–2014). Nonthaburi: Bureau of Reproductive Health, Department of Health, Ministry of Public Health, 2014.\n\nChandra-Mouli V, Svanemyr J, Amin A, et al.: Twenty years after International Conference on Population and Development: where are we with adolescent sexual and reproductive health and rights? J Adolesc Health. 2015; 56(1 Suppl): S1–6. PubMed Abstract \| Publisher Full Text\n\nDenno DM, Hoopes AJ, Chandra-Mouli V: Effective strategies to provide adolescent sexual and reproductive health services and to increase demand and community support. J Adolesc Health. 2015; 56(1 Suppl): S22–41. PubMed Abstract \| Publisher Full Text\n\nHealth Assembly: Solving Thai teen problems with pregnancy. 2016. Reference Source\n\nKuruvilla S, Bustreo F, Kuo T, et al.: The Global strategy for women’s, children’s and adolescents’ health (2016–2030): a roadmap based on evidence and country experience. Bull World Health Organ. 2016; 94(5): 398–400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLavin C, Cox JE: Teen pregnancy prevention: current perspectives. Curr Opin Pediatr. 2012; 24(4): 462–469. PubMed Abstract\n\nLeerlooijer JN, Bos AE, Ruiter RA, et al.: Qualitative evaluation of the Teenage Mothers Project in Uganda: a community-based empowerment intervention for unmarried teenage mothers. BMC Public Health. 2013; 13: 816. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeechamnan C, Fongkaew W, Chotibang J, et al.: Do Thai parents discuss sex and AIDS with young adolescents? A qualitative study. Nurs Health Sci. 2014; 16(1): 97–102. PubMed Abstract \| Publisher Full Text\n\nMueller T, Tevendale HD, Fuller TR, et al.: Teen Pregnancy Prevention: Implementation of a Multicomponent, Community-Wide Approach. J Adolesc Health. 2017; 60(3S): S9–S17. PubMed Abstract \| Publisher Full Text\n\nNitirat P: Thai adolescent’s sexual behaviors and school based sex education: perspectives of stakeholders in Chanthaburi Province, Thailand. Chapel hill: university of North Carolina at Chapel hill, 2007; 278. Reference Source\n\nPhoochaemchot P, Chomnirat W: Development Guideline to Prevent Sexual Risk Behaviors among Adolescent. Graduate Research conference 2012. Khon Kaen university. 2012; 646–657. Reference Source\n\nPoonkhum Y, Promprapat P, Paisalapachong K, et al.: The study to promote youth health and prevention of risk behaviors and health problems among youths. Nonthaburi: Bureau of Reproduction Health, Department of Health, Ministry of Public Health, 2010.\n\nSridawruang C, Crozier K, Pfeil M: Attitudes of adolescents and parents towards premarital sex in rural Thailand: a qualitative exploration. Sex Reprod Healthc. 2010a; 1(4): 181–7. PubMed Abstract \| Publisher Full Text\n\nSridawruang C, Pfeil M, Crozier K: Why Thai parents do not discuss sex with their children: a qualitative study. Nurs Health Sci. 2010b; 12(4): 437–443. PubMed Abstract \| Publisher Full Text\n\nSvanemyr J, Amin A, Robles OJ, et al.: Creating an enabling environment for adolescent sexual and reproductive health: a framework and promising approaches. J Adolesc Health. 2015; 56(1 Suppl): S7–14. PubMed Abstract \| Publisher Full Text\n\nTantisewas S, Pilasan S, Indira M, et al.: Adolescent pregnancy in Thailand 2013. Bankok: Health Intervantion and Technology Assessment Program, Ministry of Public Health, 2014.\n\nTevendale HD, Fuller TR, House LD, et al.: Implementation of Community-Wide Teen Pregnancy Prevention Initiatives: Focus on Partnerships. J Adolesc Health. 2017; 60(3S): S7–S8. PubMed Abstract \| Publisher Full Text\n\nUnited Nations Children's Fund (UNICEF): Situation analysis of adolescent pregnancy in Thailand. Synthesis report 2015. Bangkok: UNICEF Thailand, 2016. Reference Source\n\nUnited Nations Population Fund (UNFPA), Roger I: Strategic Review and Recommendations for Driving Strategies for Prevention and Prevention of Adolescent Pregnancy under the Prevention and Prevention of Adolescent Pregnancy Act. 2016. Reference Source\n\nWorld Health Organization (WHO): Health for the world’s adolescents: a second chance in the second decade. Geneva: Department of Maternal, Newborn, Child and Adolescent Health, 2014. Reference Source\n\nWorld Health Organization: Adolescent pregnancy Fact sheet. 2018. Reference Source" }	[ { "id": "32829", "date": "23 Apr 2018", "name": "Roger Ingham", "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 potentially important research that should enable exploration of the likely barriers to, and opportunities for, increasing effort to reduce unplanned pregnancies among young people in rural areas of northeast Thailand. An impressive sample size of professionals (103) was involved, although 25 to 28 in each of the groups/workshops is rather on the high side.\nWith such a large group in a workshop format, there is a risk of socially desirable responses being made, some of the participants not feeling able – or confident enough - to voice their opinions, etc.; having said this, the context does enable a wide range of views to be gathered relatively quickly.\nAlongside the group sessions some questionnaire data from young people were collected (see the supplementary material); I must confess, however, to finding the questions that were asked to be rather vague, and the response options were not specified. For example, what assumptions can be made regarding whether or not a young person has had dinner with, or enjoys nightlife with, or has a sleep over with, etc. someone from the opposite sex? Is it assumed that sex took place on these occasions? Further, a tighter translation of the instrument would assist readers.\nThe literature review is brief but highlights some of the traditional barriers to working on sexual issues with young people; some studies are from Thailand while others are from the USA and drawn from WHO overviews. It is not clear to what extent the USA studies are of relevance to the Thai context, and this could be discussed more fully. Some greater attention could be paid to justifying the value of talking to front-line professionals on this topic; they are after all an essential component of the process of implementation of any new policy initiatives in the field.\nBut, from this perspective, the results prevented are a little disappointing. It is difficult to separate out what seem to be the personal opinions of those involved in the research, rather than being professional assessments of what is required at a policy and implementation level. To be frank, little is reported that had not been reported already in other research (for example, the US research cited, and the earlier work of Chamroonsawat et al. (spelt as Chamroonsawadi in one place in the text).\nThere are some specific issue that would have benefited from greater attention and/or reporting. These include the implications of quite a high proportion of young people being brought up by grandparents – what additional barriers (over and above those present for biological parents) are imposed by this? What suggestions were made (if any) for helping to overcome these barriers?\nSecond, the phrase ‘cultural traditions and beliefs’ could benefit from unpacking further, and these workshops could have been a chance to explore these issues, whether they are still relevant, how they can be addressed, to what extent are they gendered, whether they are related to early marriage and the implications of this for birth rates, etc. Nothing is static, and change can occur – the issue is how and what is needed to speed it up?\nThe suggestions for policy are very worthy, but what is preventing them happening? Resources? Attitudes? Fear? Shifting the ‘blame’ elsewhere? Some of the suggestions are rather vague, and would benefit from greater focus. One example concerns the mention of young people having greater access to pornography. The implication made is that access should be restricted, but achieving this is highly unrealistic; so what other approaches might be helpful to get young people more engaged in consideration of gender violence, pleasure, peer and partner pressures, etc., and relate more to the fast changing social environment in which young people are growing up?\nFinally, the fieldwork for this study was carried out in 2014. Since then, there have been two national conferences on early pregnancy in Thailand, as well as the passing of an Act of Parliament on the topic (based on a rights approach). These do not get any mentions, which is regrettable given the potential relevance of the data collected in this study to implementation.\nI would encourage the authors of this article to dig somewhat deeper into the wealth of material that must have been collected from the workshops and prepare further publications to provide assistance to the challenges facing the country in the implementation of the ambitious targets for reducing the rate of early births.\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? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "35831", "date": "11 Jul 2018", "name": "Kenda Crozier", "expertise": [ "Reviewer Expertise Maternal health", "midwifery", "family 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\nThis is an interesting study and seeks to answer a very complex question which has occupied researchers in the region for some time.\nThere is a general background literature which is used to support the views of the authors which are strongly expressed in terms of the need for participation of adolescents in all stages of the planning and development of interventions. If the evidence is so strong then it would seem to indicate that this research study itself is not needed and that the development of parental and family training in communication about sexuality should be developed.\nThe methods used appear to me to be a form of realist evaluation. The paper would benefit from revisions to strengthen the methodological section and clarify the method with some recourse to the methodological literature.\nParticipants: there are a large number of participants and there is a lack of detail about the backgrounds. A table of information about the participant which groups them into health professionals, educational professionals, parents, volunteers etc would be helpful.\nThe social status of individuals within the groups may have some bearing on the way in which they interact. There is a strong sense of conformity to social hierarchy in Thailand and this may influence the way individuals feel able to express their true views. The study by Sridawruang cited in the discussion found that when parents were interviewed in focus groups they felt the need to present a publicly acceptable set of views which differed considerably to their experience expressed in one to one interviews.\nA little more detail about the workshops could shed some light on how the data emerged.\nThe nature of the findings which are unsurprising, supports the opinions of the authors cited at the outset. The problems identified are not new. The purpose of the workshops in finding potential solutions is not met unfortunately.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-428
https://f1000research.com/articles/4-1453/v1	16 Dec 15	{ "type": "Software Tool Article", "title": "MSL: Facilitating automatic and physical analysis of published scientific literature in PDF format", "authors": [ "Zeeshan Ahmed", "Thomas Dandekar" ], "abstract": "Published scientific literature contains millions of figures, including information about the results obtained from different scientific experiments e.g. PCR-ELISA data, microarray analysis, gel electrophoresis, mass spectrometry data, DNA/RNA sequencing, diagnostic imaging (CT/MRI and ultrasound scans), and medicinal imaging like electroencephalography (EEG), magnetoencephalography (MEG), echocardiography (ECG), positron-emission tomography (PET) images. The importance of biomedical figures has been widely recognized in scientific and medicine communities, as they play a vital role in providing major original data, experimental and computational results in concise form. One major challenge for implementing a system for scientific literature analysis is extracting and analyzing text and figures from published PDF files by physical and logical document analysis. Here we present a product line architecture based bioinformatics tool ‘Mining Scientific Literature (MSL)’, which supports the extraction of text and images by interpreting all kinds of published PDF files using advanced data mining and image processing techniques. It provides modules for the marginalization of extracted text based on different coordinates and keywords, visualization of extracted figures and extraction of embedded text from all kinds of biological and biomedical figures using applied Optimal Character Recognition (OCR). Moreover, for further analysis and usage, it generates the system’s output in different formats including text, PDF, XML and images files. Hence, MSL is an easy to install and use analysis tool to interpret published scientific literature in PDF format.", "keywords": [ "Bioinformatics", "Data mining", "Images", "Scientific literature", "Text", "OCR", "PDF", "Biomedical" ], "content": "Introduction\n\nThere has been an enormous increase in the amount of the scientific literature in the last decades1. The importance of information retrieval in the scientific community is well known; it plays a vital role in analyzing published data. Most published scientific literature is available in Portable Document Format (PDF), a very common way for exchanging printable documents. This makes it all-important to extract text and figures from the PDF files to implement an efficient Natural Language Processing (NLP) based search application. Unfortunately, PDF is only rich in displaying and printing but requires explicit efforts in the extraction of information, which significantly impacts the search and retrieval capabilities2. Due to this reason several document analysis based tools have been developed for physical and logical document structure analysis of this file type.\n\nThe recently, provided basic information retrieval (IR) system by PubMed is efficient in extracting literature based on published text (titles, authors, abstracts, introduction etc.), with the application of automatic term mapping and Boolean operators3. The normal outcome of a successful NLP query brings a maximum of 20 relevant results per page; however, user can improve the search by customizing the query using the provided advanced options. So far, the current PubMed system, as well many other related orthodox NLP approaches are unable to completely implement an efficient information retrieval system, capable of extracting both text and figures from published PDF files.\n\nOne of the major and technical challenges is the availability of structured text and figures. To our limited knowledge, there still is no single tool available which can efficiently perform both physical and logical structure analysis of all kinds of PDF files and can extract and classify all kinds of information (embedded text from all kinds of biological and scientific published figures). Different commercial and free downloadable software applications provide support in extracting the text and images from PDF files:\n\nA-PDF (http://www.a-pdf.com/image-extractor/),\n\nPDF Merge Split Extract (http://www.pdf-technologies.com/pdf-library-merge-split.aspx),\n\nBePDF (http://haikuarchives.github.io/BePDF/), KPDF (https://kpdf.kde.org),\n\nMuPDF (http://mupdf.com), Xpdf tool (http://www.foolabs.com/xpdf/),\n\nPower PDF (http://www.nuance.com/for-business/imaging-solutions/document-conversion/power-pdf-converter/index.htm)\n\nHowever, these software applications do not provide text and images in a form where they could be considered for further logical analysis e.g. mining text in reading order from double or multiple columns documents, searching marginal text using key-words, removing irrelevant graphics and extracting embedded text inside single and multi-panel complex biological images.\n\nSo far, the current PubMed system as well many other related orthodox NLP approaches e.g.4–13, are unable to completely implement an efficient information retrieval system, capable of extracting both text and figures from published PDF files.\n\nTo meet the technological objectives of this challenge, we took a step forward in the development of a new user friendly, modular and client based system (MSL) for the extraction of full and marginal text from PDF files based on the keywords and coordinates (Figure 1). Since MSL provides a module for the extraction of figures from PDF files and applies Optical Character Recognizer (OCR) to extract text from all kinds of biomedical and biological Images. MSL comprises three modules working in product-line architecture: Text, Image and OCR (Figure 2). Each module performs its task independently and its output is used as an input for the next module. It can be configured on Microsoft Windows platforms following a simple six-step installation process.\n\nThis figure shows the graphical user interface and modular workflow of three main components: Text, Image and OCR.\n\n\nMethods\n\nMSL extracts text and figures from the published scientific literature and helps in analyzing embedded text inside figures. The overall methodological implementation and workflow of the MSL is divided into two processes: (I) Text mining and (II) Image analysis. MSL is a desktop application, designed and developed following the scientific software engineering principles of three-layered Butterfly14 software development model.\n\nPhysical and logical document analysis is one of the living challenges. To the best of the authors’ knowledge, there is no solution available which can perform efficient physical and logical structural analysis of PDF files, implement completely correct rendering order and classify text in all possible categories e.g. Tile, Abstract, Headings, Figure Captions, Table Captions, Equations, References, Headers, Footers etc.\n\nHowever, there are some tools available which are helping in this regard e.g. PDF2HTML towards contextual modeling of logical labelling15, PDF-Analyzer for object level document analysis16, XED for hidden structure analysis2, Dolores for the logical structure analysis and recovery17 automatic conversation from PDF to XML18 and PDF to HTML19 etc.\n\nWe developed MSL’s Text module, which is capable of processing PDF files with single, double or multiple columns. It divides the system’s text based output in four sub-modules: full text, marginal text, keyword based extracted text and file attributes. Full text gives the complete text from PDF file, marginal allows user to give the coordinates (Lower Left X, Lower Left Y, Upper Right X and Upper Right Y) and extract the desired portion of the text from the PDF file. The keyword based text allows user to extract the information from PDF file based on keywords and respective coordinates (Left, Top, Width, Height) e.g. if a user is only interested in getting the figure caption or references, this kind of search will be helpful. The last sub module, File attributes gives the information about input file including title, author, creator, producer, subject, creation date, keywords, modified, number of pages and number of figures.\n\nWhile implementing Text module, we researched and tried different available commercial and freely downloadable libraries with a focus on full text extraction, marginal text extraction, keyword based text extraction and text extraction from embedded images from PDF files. We tried different implemented systems and libraries e.g. iTextSharp (http://sourceforge.net/projects/itextsharp/), Bytescout (https://bytescout.com), Spire PDF (http://www.e-iceblue.com/Introduce/pdf-for-net-introduce.html), Sautinsoft PDF Focus (http://www.sautinsoft.com/products/pdf-focus/), Dynamic PDF (https://www.dynamicpdf.com), PDFBox (https://pdfbox.apache.org), iText PDF (http://itextpdf.com), QPDF (http://qpdf.sourceforge.net), PoDoFo (http://podofo.sourceforge.net), Haru PDF Library (http://libharu.sourceforge.net), JPedal (https://www.idrsolutions.com/jpedal/), SVG Imprint (http://svgimprint-windows.software.informer.com), Glance PDF Tool Kit (http://www.planetpdf.com/forumarchive/53545.asp), BCL (http://www.pdfonline.com/corporate/) SharpPDF (http://sharppdf.sourceforge.net) etc.\n\nOne of the common problems in almost all libraries is merging and mixing of text, using double or multiple columns. Our developed system is the combination of different libraries, useful for different purposes. We have used Spire PDF to remove the Book-marks, iTextSharp for the extraction of full and marginal text, Bytescoute for the keyword based marginalized text search and producing output in the form of XML file (Figure 2). The generated XML file contains structured (tagged) text along with the information about its coordinates (placement in the file), font (Bold, Italic etc.) and size, which can be used for mapping and pattern recognition tasks.\n\nThis figure shows the conceptual architecture of the MSL application, which consists of three main components: Text, Image and OCR.\n\nImage-based analysis is a versatile and inherently multiplexed approach as it can quantitatively measure biological images to detect those features, which are not easily detectable by a human eye. Millions of figures have been published in scientific literature that includes information about results obtained from different biological and medicinal experiments. Several data and image mining solutions have been already implemented, published and are in use in the last 15 years20. Some of the mainstream approaches are towards the analysis of all kinds of images (flow charts, experimental images, models, geometrical shapes, graphs, images of thing or objects, mixed etc.). There are not many approaches proposed for specific kinds of image-analysis e.g. towards the identification and quantification of cell phenotypes21, prediction of subcellular localization of proteins in various organism22, analysis of gel diagrams23, mining and integration of pathway diagrams24.\n\nWhile implementing a new data-mining tool, one of our goals was to extract images from published scientific literature and try to extract embedded text as well. We analyzed different freely available and commercial OCR systems and libraries including Aspose, PUMA, Microsoft OCR, Tesseract, LEADTOOLS, Nicomsoft OCR, MeOCR OCR, OmniPage, ABBYY, Bytescout claiming to be able to extract embedded text from figures. During our research we found LEADTOOLS (Figure 2) as one of the best available solutions for this purpose. MSL is capable of automatically extracting images from the PDF files and allowing the user to apply OCR to any extracted image by clicking and enlarging it for a better view (using Windows default image viewer).\n\n\nDiscussion\n\nWe tested MSL with similar parameters on randomly selected scientific manuscripts (ten PDF files) from different open access (F1000Research, Frontiers, PLOS, Hindawi, PeerJ, BMC) and restricted access (Oxford University Press, Springers, Emerald, Bentham Science, ACM) publishers, including some of the authors’ published papers, details are given in Table 1. While testing MSL on the selected manuscripts, we observed best overall performance for the manuscripts26,38–42, with satisfactory results from almost all publishers (including Oxford University Press, BMC, Frontiers, PeerJ, Bentham Science, ACM) in terms of both extracting text in reading order and extracting images. An observed poor performance involved manuscripts from PLOS35, Hindawi36, F1000Research34 and IEEE37 publishers. Here, in the case of text extraction we observed that the text was in reading order when using manuscripts from F1000Research and IEEE but text was without spaces in the manuscript from PLOS and with additional lines and extra spaces in the manuscript from Hindawi. In the case of figure extraction we observed one common problem among the four manuscripts from these publishers; along with the manuscript images (Figures), embedded journal or publishers’ logos and images were also extracted. Additionally, while analyzing the manuscript from F1000Research, we observed that the images were broken into many pieces and it was not possible to find one single complete image. As we did not test all manuscripts from the mentioned publishers, we cannot claim that the results will be the same for all papers from a publisher, as the output may vary in different papers. Our observed results using MSL are given in attached supplementary material (Supplementary Table S1 and Dataset 1).\n\nThe table gives the list of 10 of those manuscripts from different publishers, which have been used for testing and validating the MSL application.\n\nTo apply MSL, published scientific literature has first to be downloaded in the form of a PDF file, from any published source. The validation process using MSL consists of three major steps: 1) Text mining, 2) Image extraction, and 3) Application of OCR to extract text from selected images as shown in Figure 1, following the implemented workflow as shown in Figure 2. Example results and graphics are shown in Figure 1, Figure 3 and Figure 4. Representation includes the extraction of text and images from one of the randomly selected papers25, and application of OCR to one of the extracted images from another randomly picked publication26.\n\nThis figure shows document image analysis, text extraction and PDF conversion.\n\nThis figure shows document image analysis, text extraction and PDF conversion.\n\nFigure 1 shows that one randomly selected published article’s PDF file25 is inputted to the MSL’s text, the extracted text is divided into three categories (i) complete text in excellent rendering order (ii) marginalized text and (iii) keyword based searched text. Two figures (Figure 1 and Figure 2) are extracted and displayed in the image section, and one of those is selected to apply OCR. The applied OCR extracts textual information, which is displayed in and can be exported in a PDF file.\n\nTo further validate the application of OCR and discuss different results, Figure 3 show another example of embedded text extraction from a complex figure27, which includes three panels of images (i) colorful pie and circle charts, (ii) biological images and (iii) tabular information. Similar to our prior application of OCR, results are displayed in textual form as well as generated PDF file of extracted text. A noticeable difference between both outputs is that the textual information is presented in line-by-line order whereas in the PDF file the information is displayed in margins with respect to the original image.\n\nThe last resultant example is based on the validation of MSL by extracting the textual information from image based PDF files. We produced an image form of one of the randomly selected article26 and then processed one of pages. As Figure 4 shows, the obtained results were comprehensive in both textual as well as the PDF form. This kind of textual extraction can be very helpful, especially when the literature is available in only images e.g. in the case of old published literature in print only format but electronically available in scanned form. MSL produces several files as system output in the parent folder of the files. These files are: XML files (which include structured or tagged information), an Images File (extracted from the PDF file) and PDF files for all analyzed images using OCR.\n\nWe mentioned earlier that we have tried and implemented different libraries for text and image extraction and analysis. The best text based outcome was observed using iTextSharp, better image extraction was observed using Spire and OCR from LEADTOOLS was the most promising. While validating the implemented solution, other than the expected results (text and images), we observed some limitations in the used libraries: unexpected and irrelevant images were also extracted e.g. journal, publisher’s logos and header-footer images, text was not always in good rendering order, especially when there were text-based mathematical equations with super and subscripts; and in case of double or multicolumn PDF files, most of the libraries’ rendering order is not correct. During extracting text, we found that some important symbols were missed and spaces were generated for some paragraphs. We found that it was not possible to extract particular images that are created as a combination of different sub-images and text objects in the manuscript. In these cases, text is found in extracted text area and all extracted sub-images are image sections, with the possibility of missing some sub-images as well. Moreover, when we applied OCR to different images (extracted or loaded), we found that its performance does vary with respect to the complexity of inputted images. In case of special characters (e.g. Greek delta, alpha, beta etc.), it does not perform well unless these are hard wired in the software.\n\nTo enhance the functionality of the MSL program (e.g. our standard version available here for download), we give a table of the most often used special symbols in biomedical literature (Table 2). Depending on your application in mind, you thus simply extend the MSL parser by considering also these special characters occurring often in your texts.\n\n1The table illustrates that special characters occurring most often in the texts of choice enhance further MSL capabilities if incorporated in addition in the parser. This is, however, a text-dependent additional modification of the MSL program\n\n\nImplementation & operation\n\nMSL architecture is based on the Product Line Architecture (PLA) and Multi-Document Interface (MDI) developmental principles, and it is designed and developed (using C-Sharp programming language, Microsoft Dot NET Framework) following the key principles of Butterfly paradigm14,27. The work-flow of MSL is divided into two processes: (I) extraction and marginalization of text with respect to the division and placement of text in PDF file and keyword based search by using the iTextSharp, Bytescoute, Spire PDF libraries, and (II) extraction and analysis of figures by using the Spire PDF library and LEADTOOLS OCR.\n\nIt takes Portable Document Format (PDF) based literature files as input, performs partial physical structure analysis, and exports output in different formats e.g. text, images and XML files. It allows user to extract keywords and marginal (X and Y coordinates) information based text, have PDF file’s metadata information (title, author, creator, producer, subject, creation date, keywords, modified, number of pages and number of figures) and save extracted full and marginal text in text files.\n\nBiomedical image extraction and analysis is one of the most complex tasks from the field of computer sciences and image analysis. Some of the mainstream approaches28–33 have been proposed towards the analysis of all kinds of images (e.g. flow charts, experimental images, models, geometrical shapes, graphs, image-of-thing, mix etc.). MSL allows user to automatically extracting images from the PDF files, let any selected image viewed via Windows default image viewer and apply implemented OCR. Other than extract images from PDF file, MSL allow user to load any image, apply OCR and export output in readable PDF file.\n\nMSL produces several out files in the parent folder including XML files (which include structured or tagged information), Images File (extracted from PDF file) and PDF files for all analyzed images using OCR (Figure 5).\n\nMSL application is very simple to install and use. It was tested and can be well configured on a Microsoft Windows platform (preferred OS version: 7). MSL follows a simple six steps installation process (Figure 6). After installation, it can be run by either clicking on the installed application’s icon at the desktop or execute application following sequence of steps: Start → All Programs → MSL 1.0.0 → MSL.\n\nRegarding using the MSL application, one important point to remember is that it is based on different PDF text extraction, marginalization and figure extraction libraries, which are automatically configured during installation but used OCR by the LEADTOOLS is not a freely available library, which we have used upon academic research (free) license. The OCR library is also automatically configured during installation but its performance at different (non-licensed) machines is not confirmed. Moreover, the recommended display screen resolution size is 1680×1050 with landscape orientation.\n\n\nConclusions\n\nThe latest available and easy to use version of MSL has been tested and validated in-house. The advancements in information retrieval techniques for text and figure analysis combined with this sophisticated computational tool can support various studies. As future work, we are looking forward to further implement machine learning and pattern recognition methods for the extracted text classification.\n\n\nData availability\n\nF1000Research: Dataset 1. Extracted Images and Text from Papers tested using MSL, 10.5256/f1000research.7329.d10873943\n\n\nSoftware availability\n\nThe software executable is freely available at the following web link: https://zenodo.org/record/30941#.Vi0PtmC5LHM\n\nThe software download section provides one executable: MSL, setup to be installed on the Microsoft Windows platform.\n\nMSL has been NOT been developed for any commercial purposes but as a non-commercial prototype application for academic research, analysis and development purposes.\n\nMining Scientific Literature (MSL) Ver 1.0.0 (DOI: 10.5281/zenodo.30941).\n\nAll associated files are licensed under the Academic Free License 3.0 (AFL 3.0).", "appendix": "Author contributions\n\n\n\nZA: developed the complete solution (including research, software designing, programming, testing, deployment and technical documentation). TD guided the study. All authors participated in writing of the manuscript and approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by a German Research Foundation grant (DFG-TR34/Z1) to TD.\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 German Research Foundation (DFG-TR34/Z1) for support. We would like to thank Dr. Chunguang Liang (University of Wuerzburg, Germany) for his help in testing MSL and all interested colleagues for critical community input on the approach and anonymous reviewers for their helpful comments.\n\nWe would like to thank all the open source, licensed and commercial library providers, for their help in this non-commercial and academic research and software development.\n\n\nSupplementary material\n\nSupplementary Table S1. List of Papers (PDF files) tested using MSL\n\nSupplementary which gives the list of some of those manuscripts from different publishers (F1000Research, PLOS, Hindawi, IEEE, BMC, PeerJ, Frontiers, ACM, Bentham Science and Oxford University Press), which have been used for testing and validating the MSL application. The attached table provides the information about some of the extracted images and observed full and marginal text.\n\nClick here to access the data.\n\n\nReferences\n\nHunter L, Cohen KB: Biomedical language processing: what’s beyond PubMed? Mol Cell. 2006; 21(5): 589–594. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHadjar K, Rigamonti M, Lalanne D, et al.: Xed: A New Tool for Extracting Hidden Structures from Electronic Documents. In International Workshop on Document Image Analysis for Libraries. 2004; 221–224. Publisher Full Text\n\nSayers EW, Barrett T, Benson DA, et al.: Database resources of the National Center for Biotechnology Information. 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PubMed Abstract \| Publisher Full Text\n\nXu YY, Yang F, Zhang Y, et al.: Bioimaging-based detection of mislocalized proteins in human cancers by semi-supervised learning. Bioinformatics. 2015; 31(7): 1111–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhmed Z, Zeeshan S: Cultivating Software Solutions Development in the Scientific Academia. Recent Patents on Computer Sci. 2014; 7(1): 54–66. Publisher Full Text\n\nSchindelin J, Arganda-Carreras I, Frise E, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmid B, Schindelin J, Cardona A, et al.: A high-level 3D visualization API for Java and ImageJ. BMC Bioinformatics. 2010; 11: 274. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(7): 671–75. PubMed Abstract \| Publisher Full Text\n\nPeng H, Ruan Z, Long F, et al.: V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets. Nat Biotechnol. 2010; 28(4): 348–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLopez LD, Yu J, Arighi C, et al.: A framework for biomedical figure segmentation towards image-based document retrieval. BMC Syst Biol. 2013; 7(Suppl 4): S8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSheng J, Xu S, Deng W, et al.: Novel Image Features for Categorizing Biomedical Images. In IEEE International Conference on Bioinformatics and Biomedicine (BIBM). 2012. Publisher Full Text\n\nAhmed Z, Zeeshan S, Fleischmann P, et al.: Ant-App-DB: a smart solution for monitoring arthropods activities, experimental data management and solar calculations without GPS in behavioral field studies [version 3; referees: 2 approved, 1 approved with reservations]. F1000Res. 2015; 3: 311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPryszcz LP, Németh T, Saus E, et al.: The Genomic Aftermath of Hybridization in the Opportunistic Pathogen Candida metapsilosis. PLoS Genet. 2015; 11(10): e1005626. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHernández JC, Rodríguez JM, Sigarreta JM: Mathematical Properties of the Hyperbolicity of Circulant Networks. Advances in Mathematical Physics. 2015; 2015: 11. Publisher Full Text\n\nZeeshan A, Detlef G: Design implementation of I-SOAS IPM for advanced product data management. IEEE 2nd International Conference on Computer, Control and Communication. 2009; 1–5Publisher Full Text\n\nAhmed Z, Zeeshan S, Huber C, et al.: Software LS-MIDA for efficient mass isotopomer distribution analysis in metabolic modelling. BMC Bioinformatics. 2013; 14: 218. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEren AM, Esen ÖC, Quince C, et al.: Anvi'o: an advanced analysis and visualization platform for 'omics data. PeerJ. 2015; 3: e1319. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoreau T, Gibaud B: Ontology-based approach for in vivo human connectomics: the medial Brodmann area 6 case study. Front Neuroinform. 2015; 9: 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhmed Z: Intelligent semantic oriented agent based search (I-SOAS). In Proceedings of the 7th International Conference on Frontiers of Information Technology. 2009. Publisher Full Text\n\nAhmed Z, Helfrich-Förster C: DroLIGHT-2: Real Time Embedded and Data Management System for Synchronizing Circadian Clock to the Light-Dark Cycles. Recent Patents on Computer Sci. 2013; 6(3): 191–205. Publisher Full Text\n\nAhmed Z, Dandekar T: Dataset 1 in: MSL: Facilitating automatic and physical analysis of published scientific literature in PDF format. F1000Research. 2015. Data Source" }	[ { "id": "11637", "date": "13 Jan 2016", "name": "Juilee Thakar", "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 manuscript titled “MSL: Facilitating automatic and physical analysis of published scientific literature in PDF format” addresses an important issue of extracting information from published manuscripts. However, the following issues must be clarified before indexing.In the text mining section authors say that there is no tool to perform physical and logical structural analysis of PDF files. However, in the next paragraph they describe “Dolores” for logical structure analysis. Authors should describe how their method is different than Dolores.Legends of all the figures should be more descriptive so that figures are understandable on their own. Each component of the figure should be described in the legend.The results section is missing. Is it integrated in the discussion section? It is unclear what exactly the results were.The article will be much clear if all the libraries (described on page 4 second paragraph) are described in the form of a table.Authors should include a clear metric to estimate performance of the algorithm. This can be achieved by comparison with existing tools or through comparative analysis. A clear example showing the information extracted from several PDF files to address a biologically relevant example will be useful.It is not clear whether the text extracted from the PDF files is actually coming from figure legends or related to the main body of the manuscript. Also, how is this text organized?The authors mention that unexpected and irrelevant images were extracted. It is not clear how authors address that. It is absolutely essential to address that.Minor corrections:Page 2 second column: The definition of MSL is not the same as described in the abstract", "responses": [ { "c_id": "2627", "date": "12 Apr 2017", "name": "Zeeshan Ahmed", "role": "Reader Comment", "response": "Reply: Thank you so much for your recommendations. The manuscript titled “MSL: Facilitating automatic and physical analysis of published scientific literature in PDF format” addresses an important issue of extracting information from published manuscripts. Reply: Thanks. However, the following issues must be clarified before indexing. Reply: Sure. In the text mining section authors say that there is no tool to perform physical and logical structural analysis of PDF files. However, in the next paragraph they describe “Dolores” for logical structure analysis. Authors should describe how their method is different than Dolores. Reply: Thanks for the nice suggestion. We have provided a brief comparison with Dolores, as well as some other mentioned tools in the paper. This includes Dolores, PDF2HTML, XED, and PDF-Analyzer, too and is now mentioned first time when mentioning Dolores. Legends of all the figures should be more descriptive so that figures are understandable on their own. Each component of the figure should be described in the legend. Reply: Thanks for pointing this out, we have revised the manuscript and added more details to the figure legends explaining the symbols used. The results section is missing. Is it integrated in the discussion section? It is unclear what exactly the results were. Reply: Yes, we have an integrated results and discussion section, please accept apologies for this confusion. To further clarify it, we have revised the heading titles and stressed the results achieved and subsequently discussed by subtitles. The article will be much clear if all the libraries (described on page 4 second paragraph) are described in the form of a table. Reply: Thanks for this important suggestion. We have added a table to the manuscript showing all libraries we tested for MSL as well as whether they are partly or completely integrated in MSL. (i) Authors should include a clear metric to estimate performance of the algorithm. This can be achieved by comparison with existing tools or through comparative analysis. (ii) A clear example showing the information extracted from several PDF files to address a biologically relevant example will be useful. Reply: Thanks for these valuable suggestions. (i). Initially, we also aimed to perform such comparative analysis, but most of the used and tested libraries are from different commercial and licensed sources, and algorithmic details were not given. We choose to go for commercial and licensed libraries because open source libraries and methods (we found) were unable to meet the developmental objectives this software. Without such details it is not possible to draw comparative (algorithmic, metrics based) conclusions. However, we give now a feature-based comparison in the results and discussion section of the manuscript. This also clearly shows the advantages of the MSL software. Furthermore, we discuss also the limitations and possible extensions of our MSL software, again referring to other existing software. (ii): We have added a detailed example in the manuscript and some further examples of text and image extraction to the supplementary material. Furthermore, we have added performance details, these highlight the biological importance as well. It is not clear whether the text extracted from the PDF files is actually coming from figure legends or related to the main body of the manuscript. Also, how is this text organized? Reply: Please accept apologies for this confusion. The extracted text from PDF files is coming from the main text as well as the figure legends. The text is organized in reading order e.g. in the case of a two or multiple columns document; the text of the first column is followed by the text of the second column, and so on. The authors mention that unexpected and irrelevant images were extracted. It is not clear how authors address that. It is absolutely essential to address that. Reply: We apologize to have caused here some confusion. The “irrelevant images” are e.g. journal, publisher’s logos and header-footer images embedded inside document. These images are often added by the publishers of journal or conference, which have nothing to do with the actual manuscript’s content but to clear to the reader about publication details. We explain this in the manuscript including how these are removed. Minor corrections: Page 2 second column: The definition of MSL is not the same as described in the abstract. Reply: Thanks for pointing this out; we have corrected this and are now more accurate when introducing MSL on page 2." }, { "c_id": "3586", "date": "11 Apr 2018", "name": "Juilee Thakar", "role": "Reviewer Response", "response": "Thanks for responding to my suggestions." } ] }, { "id": "14625", "date": "09 Aug 2016", "name": "M. Julius Hossain", "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 authors presented a computational tool that extracts text and images from PDF files. In general the manuscript is interesting considering that it can analyze various types of PDF files from different scientific areas based on the keywords and coordinates. However, it lacks technical novelty over the published literatures and needs additional input on the image analysis section before indexing.\nExtraction of texts and images from scientific publications has been presented in various domains: computer science1, biomedical2-4, chemistry5, proteomics6 and so on. The manuscript by Zeeshan Ahmed and Thomas Dandekar presents an incremental innovation without providing clear technological advancement in the field. The objective of performing both physical and logical structure analysis of all kinds of PDF files as mentioned in the manuscript has not been sufficiently supported by technological contribution described in Methods section.\nThe image processing section the manuscript has been very brief. It does not provide any advanced image analysis technique as mentioned in the abstract. Authors should mention how exactly segmentation of figures and labels are performed and how they are represented to make logical connection between different entities in order to perform further analysis and customized visualization.\nThe framework has been tested with a very small set of PDF files and no qualitative/quantitative result reporting the accuracy with respect to manually annotated files was presented. It would be good to increase the number test files and include the results of qualitative/quantitative analysis.\nSome of the figures (Figures 1, 3, 4 and 6) in the manuscript are hard to see the details in both online and print format. These figures could be reformatted.", "responses": [ { "c_id": "2628", "date": "12 Apr 2017", "name": "Zeeshan Ahmed", "role": "Reader Comment", "response": "Reply: Thank you so much for your recommendations. In this manuscript authors presented a computational tool that extracts text and images from PDF files. In general the manuscript is interesting considering that it can analyze various types of PDF files from different scientific areas based on the keywords and coordinates. Reply: Thanks. However, it lacks technical novelty over the published literatures and needs additional input on the image analysis section before indexing. Reply: Thanks for raising this point, we have revised and tried to make it more clearer. Extraction of texts and images from scientific publications has been presented in various domains: computer science1, biomedical2-4, chemistry5, proteomics6 and so on. The manuscript by Zeeshan Ahmed and Thomas Dandekar presents an incremental innovation without providing clear technological advancement in the field. The objective of performing both physical and logical structure analysis of all kinds of PDF files as mentioned in the manuscript has not been sufficiently supported by technological contribution described in Methods section. Reply: Thanks for raising this point and please accept apologies for the confusion. Its true that there have been many efforts from the past towards the same problem, and from different disciplines. We have taken a facile step by combining some of available technologies and tried to present a method, which can be adapted and further enhanced. To make our point more clearer, we have revised manuscript. The image processing section the manuscript has been very brief. It does not provide any advanced image analysis technique as mentioned in the abstract. Authors should mention how exactly segmentation of figures and labels are performed and how they are represented to make logical connection between different entities in order to perform further analysis and customized visualization. Reply: Thanks for raising this point and please accept apologies for the confusion. We agree with your point. We didn’t discuss and go in to details of algorithmic image presenting because we didn’t implement any algorithm for this work but tested and adopted some pre-existing OCR based libraries. We choose to go for commercial and licensed libraries because open source libraries and methods (we found) were unable to meet the developmental objectives this software. Without such details it is not possible to draw comparative (algorithmic, metrics based) conclusions. However, we give now a feature-based comparison in the results and discussion section of the manuscript. The framework has been tested with a very small set of PDF files and no qualitative/quantitative result reporting the accuracy with respect to manually annotated files was presented. It would be good to increase the number test files and include the results of qualitative/quantitative analysis. Reply: Thanks for raising this point and we have revised with additional details. We tested our system with similar parameters on randomly selected scientific manuscripts (ten PDF files) from different open access ( F1000Research, Frontiers, PLOS, Hindawi, PeerJ, BMC) and restricted access ( Oxford University Press, Springers, Emerald, Bentham Science, ACM) publishers. Some of the figures (Figures 1, 3, 4 and 6) in the manuscript are hard to see the details in both online and print format. These figures could be reformatted. Reply: Thanks for raising this point and we have revised Figures." } ] } ]	1	https://f1000research.com/articles/4-1453
https://f1000research.com/articles/7-426/v1	04 Apr 18	{ "type": "Research Article", "title": "Cytoglobin, neuroglobin, and acetylcholinesterase activity in rat brain as adaptation responses to intermittent hypobaric hypoxia", "authors": [ "Angelina Stevany Regina Masengi", "Fanny Septiani Farhan", "Wawan Mulyawan", "Mohamad Sadikin", "Ninik Mudjihartini", "Sri Widia A. Jusman", "Angelina Stevany Regina Masengi", "Fanny Septiani Farhan", "Wawan Mulyawan", "Mohamad Sadikin", "Ninik Mudjihartini" ], "abstract": "Background: Intermittent hypobaric hypoxia is suggested to possess a protective effect toward the hypoxic condition. The aim of this study is to analyze the expression of cytoglobin (Cygb), neuroglobin (Ngb) and the specific activity of acetylcholinesterase (AChE) in brain tissue as adaptive responses to intermittent hypobaric hypoxia. Methods: Twenty-five adult Sprague-Dawley male rats were divided into 5 groups: 1) The control group (normoxia); 2) group exposed to acute hypobaric hypoxia (AHH); 3) group exposed to hypobaric hypoxia (HH) on day-1 and re-exposed on day-8 (intermittent hypobaric hypoxia once or IHH1x); 4) group that is exposed to HH on day-1, re-exposed to HH on day-8 and day-15 (intermittent hypobaric hypoxia two times or IHH2x); 5) group exposed to HH on day-1, re-exposed to HH on day-8, day-15 and day-22 (intermittent hypobaric hypoxia 3x or IHH3x). Homogenized brain tissue was then measured and analyzed for Cygb and Ngb protein expression, and also AChE specific activity. Results: Cytoglobin and Ngb were decreased in the acute induction and increased significantly along with the increasing frequency of the IHH induction. There were significant differences in Cygb expression between IHH2x and IHH3x groups compared to normoxia group, and between IHH1x, IHH2x and IHH3x compared to AHH group. There were significant differences in Ngb expression between IHH2x and IHH3x groups compared to normoxia group and between IHH2x and IHH3x groups compared to AHH group. The specific activity of AChE was increased significantly since the first induction of AHH, but then decreased in IHH3x. There were significant differences in the specific activity of AChE between IHH2x and IHH3x groups compared to normoxia and between IHH2x and IHH3x groups compared to IHH1x groups. Conclusions: We conclude that IHH, especially IHH3x, seems to induce the protective adaptive response in the rat brain tissue through the changes of these three parameters.", "keywords": [ "cytoglobin", "neuroglobin", "acetylcholinesterase", "brain", "intermittent hypobaric hypoxia" ], "content": "Introduction\n\nCytoglobin (Cygb) and neuroglobin (Ngb) are globin proteins, found recently, which have special roles such as for oxygen supply in hypoxia. Both Cygb and Ngb have a high affinity for oxygen. These two globin proteins, which expressed by the neuron, are also known to act as scavengers of reactive oxygen species (ROS) and reactive nitrogen species1. Increase in Cygb and Ngb occurs in hemorrhagic stroke patients with increased ROS. Therefore, it is suggested that an increase in Cygb and Ngb are adaptation responses to increase oxygen supply and scavenge ROS due to hypoxia in hemorrhagic stroke2,3. Increased expression of Cygb also occurs after chronic systemic normobaric hypoxia induction in a rat's brain. An increased level of HIF-1α occurs along with the increased level of Cygb until day-7 of the induction, while the expression of Ngb was decreased in day 1, 3, 5, 7, and 14 of induction compared to normoxia4.\n\nDecreasing the level of oxygen causes energy production depletion needed by the cells for their optimal physiological function, such as a cognitive function of the brain. The cholinergic system is known to form synapses throughout the brain, including the cerebral cortex, which is part of the cognitive signaling pathway. Cholinergic signaling is terminated, among other things, by the work of acetylcholinesterase (AChE), which catalyzes the hydrolysis of ACh in the synaptic cleft to an inactive form, choline and acetic acid5.\n\nIn systemic chronic normobaric hypoxia, there was an increase of AChE specific activity, which proves that the induction is able to trigger an increase of the enzyme activity, so that it can reduce the cholinergic system function6. In an animal study, bilateral common carotid artery occlusion caused chronic cerebral hypo perfusion. At week 4, the level of ACh in the hippocampus decreased. ACh reduction also occurred in the striatal area, and was suspected to be the cause of memory consolidation disturbance that leads to learning dysfunction7.\n\nMulyawan's research has shown that HIF-1α expression was increased after intermittent hypobaric hypoxia induction in a rat using hypobaric chamber8. Until now, there is no data of how IHH affects Cygb and Ngb expression, which play a role in oxygen supply, and how this affects AChE specific activity, which plays a role in ACh, a neurotransmitter important for cognitive function, termination. The purpose of this study is to determine the effect of IHH toward these three parameters in the brain adult male of Sprague-Dawley rats.\n\n\nMethods\n\nAll procedures were approved by the Ethics Committee of the Faculty of Medicine Universitas Indonesia Rumah Sakit Cipto Mangunkusumo No. 626/UN2.F1/ETIK/2014. All efforts were made to ameliorate any suffering of animals used in this research. The rats were observed daily to confirm their healthy condition. The behavior tests, done by Farhan[18], and the hypoxia induction did not involve painful stimuli.\n\nBased on Federer’s rule, with five groups of induction, twenty-five adult Sprague-Dawley male rats from Badan Pengawasan Obat dan Makanan Republik Indonesia (BPOM RI), initially weighing 200–250 grams, were randomly divided into 5 groups: 1) The control group (normoxia); 2) group exposed to acute hypobaric hypoxia (AHH, control to intermittent hypobaric hypoxia (HH) treatment); 3) group exposed to HH on day-1 and re-exposed on day-8 (intermittent hypobaric hypoxia once, IHH1x); 4) group exposed to HH on day-1, re-exposed to HH on day-8 and day-15 (intermittent hypobaric hypoxia 2 times, IHH2x); 5) group exposed to HH on day-1, re-exposed to HH on day-8, day-15 and day-22 (intermittent hypobaric hypoxia three times, IHH3x).\n\nBefore the experiment all rats were kept under optimal conditions: 12:12 hours light to dark cycle at 24°C, in the animal house of Indonesian Air Force Institute of Aviation Medicine, LAKESPRA dr. Saryanto, Jakarta, with food and water ad libitum. Pelleted food and tap water were given. Wire cages were used to ensure optimum ventilation; subjects in a group were put in the same cage. Shredded newspaper were used and changed daily.\n\nAfter treatment, rats were euthanized by cervical dislocation. Then the brains were immediately removed, weighed and divided into aliquots and stored in -80°C.\n\nThe procedure of hypobaric hypoxia is referred to hypobaric hypoxia type I chamber flight profile, modified by Mulyawan, presented in Figure 18.\n\nProduced and previously presented by the first author at The 1st Asian Researcher Symposium at the Universitas Indonesia-Depok, April 26th 2016.\n\nIn this procedure, starting around 09.00 AM, the rats were put in a hypobaric chamber at the Aerophysiology Department of Indonesian Air Force Institute, LAKESPRA dr. Saryanto, Jakarta, and exposed to treatment ascending with rising speed of 5,000 feet/ minute until reach the altitude which equal to 35,000 feet above sea level for 1 minute, then descending gradually to 30,000; 25,000; 18,000 feet for 3; 5; and 30 minutes respectively. And then descend gradually until reaching the sea level. All descending speed is performed at 5,000 feet/ minute8.\n\nBrain tissue was homogenized using RIPA Lysis Buffer (Santa Cruz ®, product number sc-24948) before the measurement of the total protein, Cygb and Ngb proteins, using ELISA method and specific activity of AChE. These activities were conducted at the Laboratory of Molecular Biology for Oxidative Stress Studies, Department of Biochemistry and Molecular Biology, Faculty of Medicine Universitas Indonesia, Salemba, Jakarta.\n\nTotal protein concentration is determined using bovine serum albumin (BSA) as standard solution (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg/dL) and absorbance was read at λ 280 nm using Nano drop Varioskan Flash (Thermo Scientific).\n\nCygb protein concentration was measured using ELISA method. The microplate provided in the kit has been pre-coated with Cygb specific antibody. The standard solutions of Cygb were made as follows: 0 (blank); 0.78; 1.56; 3.12; 6.25; 12.5; 25; and 50 ng/mL. The absorbance was read at λ 450 nm. The concentration of Cygb was measured using Cygb standard curve. The concentration of Cygb in brain tissue was presented as the concentration of Cygb/total brain protein concentration (ng/mg protein).\n\nNgb protein concentration is determined using ELISA method. Ngb specific antibody has been pre-coated onto the microplate. The standard solutions of Ngb were made as follows: 0 (blank); 15.6, 31.2, 62.5, 125, 250, 500, and 1000 ng/dL. The absorbance was read at λ 450 nm. The concentration of Ngb was measured using Ngb standard curve. The concentration of Ngb in brain tissue was presented as the concentration of Ngb/total brain protein concentration (ng/mg protein).\n\nIn this assay, thiocholine as the product of AChE is reacted to 5,5'-dithiobis (2-nitrobenzoic acid, DTNB) to form a yellowish color product, which provides absorption at λ 412 nm. The intensity of the yellowish color is equivalent to the activity of AChE. One unit of AChE is a number of enzymes that catalyze the production of 1.0 µmol tiocholine/minute at pH 7.5 at room temperature. The specific activity of AChE was measured by dividing the AChE activity with the total protein of each sample (U/mg protein).\n\nGraph Pad Prism version 7.0 was used for statistical analysis. Data normality was examined using D’Agostino and Pearson normality test. Then data were analyzed using ANOVA for significance and further post hoc with Tukey test. The Pearson correlation test was used to examine the correlation between parameters.\n\n\nResults\n\nThe concentration of Cygb in brain tissue after induction of AHH, IHH1x, IHH2x, IHH3x, and normoxic groups, is shown in Figure-2. In this figure, we can see that there were significant differences between the IHH2x and IHH3x groups compared to normoxic group (p=0.015 and p=0.000, respectively). There was also a significant difference between IHH1x, IHH2x and IHH3x compared to the AHH group (p=0.02, p=0.001 and p=0.000, respectively). Differences also appeared between IHH1x and IHH3x (p = 0.005).\n\nAHH, control to intermittent hypobaric hypoxia treatment; IHH1x, intermittent hypobaric hypoxia once; IHH2x, intermittent hypobaric hypoxia 2 times; IHH3x, intermittent hypobaric hypoxia three times. p<0.05.\n\nIt is shown in Figure-3 that there are significant differences between the IHH2x and IHH3x groups compared to normoxic group (p=0.049 and p=0.03, respectively). There were also significant differences between IHH2x and IHH3x groups compared to the AHH group (p = 0.005 and p=0.000, respectively). Differences were also showed between IHH2x and IHH3x groups compared to IHH1x group (p = 0.022 and p=0.001, respectively).\n\nAHH, control to intermittent hypobaric hypoxia treatment; IHH1x, intermittent hypobaric hypoxia once; IHH2x, intermittent hypobaric hypoxia 2 times; IHH3x, intermittent hypobaric hypoxia three times. p<0.05.\n\nAs can be seen in Figure 4, there were differences in the specific activity of AChE in brain tissue of rats in IHH2x and IHH3x groups compared to normoxic group (p=0.000 and p=0.000, respectively). There were also significant differences between IHH2x and IHH3x with the AHH group (p=0.000 and p=0.000, respectively). There were also significant differences between the IHH2x and IHH3x groups compared to IHH1x group (p=0.000 and p=0.000, respectively), and between IHH2x and IHH3x (p=0.026).\n\nAHH, control to intermittent hypobaric hypoxia treatment; IHH1x, intermittent hypobaric hypoxia once; IHH2x, intermittent hypobaric hypoxia 2 times; IHH3x, intermittent hypobaric hypoxia three times. *p<0.05.\n\nThere was a strong positive correlation between the Cygb protein and specific activity of AChE in brain tissue of normoxic, AHH and IHH groups (Pearson, r = 0.728 and p<0.0001) (Figure-5a).\n\n(a) Correlation between Cygb protein and AChE specific activity in brain tissue of rats (Pearson; r=0.728; p<0.0001). (b) Correlation between Ngb protein AChE specific activity in brain tissue of rats (Pearson; r=0.542; p<0.0001). (c) Comparison of Cygb, Ngb protein and specific activity of AChE in brain tissue of rats. AHH, control to intermittent hypobaric hypoxia treatment; IHH1x, intermittent hypobaric hypoxia once; IHH2x, intermittent hypobaric hypoxia 2 times; IHH3x, intermittent hypobaric hypoxia three times.\n\nThere was a moderate positive correlation between the Ngb protein and specific activity of AChE in brain tissue of normoxic, AHH and IHH groups (Pearson, r = 0.542 and p<0.0001) (Figure-5b).\n\nComparison of the expression patterns between Cygb, Ngb levels and the specific activity of AChE is shown in Figure-5c. It appears that Cygb and Ngb expressions were decreased while the specific activity of AChE was increased at AHH. At the induction of IHH1x, Cygb and Ngb expressions were increased, so did the specific activity of AChE. Induction of IHH2x caused an increase in Ngb and Cygb levels and also the specific activity of AChE. At the induction of IHH3x, Cygb and Ngb expressions were increased, however, the specific activity of AChE was decreased.\n\n\nDiscussion\n\nIn this research, Cygb and Ngb expressions of AHH group were decreased compared to normoxia. Those two protein expressions were then increased in intermittent induction of hypoxia, from IHH1x induction until IHH3x. The low expressions of Cygb and Ngb in AHH group might be due to the role of these proteins in scavenging ROS produced in this induction. In repeated hypobaric hypoxia induction (IHH1x, IHH2x, and IHH3x), the increase of Cygb and Ngb expressions might be due to adaptation response toward hypoxia and the increased ROS in this condition. Cygb and Ngb are known to be increased in supplying oxygen and reducing or scavenging ROS in hypoxic conditions9–11.\n\nThe increase of specific activity of AChE was seen from AHH induction and continued to increase until IHH2x and then decreased in IHH3x. The increased specific activity of this enzyme was also reported by Muthuraju et al. in hypobaric hypoxia and Andriani et al. in chronic normobaric hypoxia6,11. An increase in AChE activity, which then caused the decrease of ACh concentration, in and around amyloid β-peptide (Aβ) (a substance which found to be increased in Alzheimer’s disease) has also been shown12. The increased activity of AChE which causes the decrease amount of ACh eventually caused a reduction in blood flow. The reduction of blood flow due to the decreased levels of ACh is due to the function of ACh in causing vasodilatation13. In brain hypoperfusion by ligating common carotid artery, there is a decrease in ACh in the hippocampal area, which also related to memory and learning impairment7. In addition, the loss of perivascular cholinergic terminals was shown in AD patients compared to aged controls14. This research was supported by other researchers who observed impaired cortical cerebral blood flow in patients with AD15.\n\nMoreover, AChE inhibitor medication is known to affect cholinergic function in subjects treated with hypobaric hypoxia (decrease acetylcholinesterase activity, increase acetylcholine levels and upregulation of choline acetyltransferase—an enzyme that has a role in acetylcholine formation) and eventually memory function11. Inhibitors of AChE are also known to improve cerebrovascular function16. This medication is known to overcome the cognitive function impairment in Alzheimer’s disease17. As a comparison in cognitive function, Farhan reports that IHH, using the same procedure with our study, increases cognitive function compared to control group18.\n\nIn this research, the specific activity of AChE was increased in AHH conditions, which meant that there was a decrease in blood flow to the brain due to the decrease of ACh. This condition may lead to a decrease in blood flow, inducing the adaptation response through increased expressions of Cygb and Ngb since IHH1x until IHH3x to increase blood/oxygen supply. However, since the blood flow was not measured in this research, this limitation needs further research to ensure whether this hypothesis is correct. Meanwhile, in IHH3x there was a decrease in AChE specific activity. It suggests that in IHH3x the blood/oxygen flow is sufficient due to the increase expressions of Cygb and Ngb which cause an increase in oxygen availability. This phenomenon is supported by Mulyawan’s research that reports an increase in microvascular density using GLUT-1 as the marker from IHH1x until IHH3x induction8. However, the mechanism of the increase of AChE activity in hypoxia requires further study.\n\nThe present research has demonstrated that intermittent hypoxia (IHH1x until IHH3x) can provide protection against hypoxia, which was shown in the increase of Cygb and Ngb that supply oxygen. Overall, this research could be implemented for individual training to adapt to hypoxia conditions, for example, air force pilot.\n\n\nConclusions\n\nWe conclude that IHH seems to induce a protective adaptive response in the rat brain tissue through the changes of Cygb and Ngb expression and the changes of AChE specific activity. Further research is needed to measure and evaluate the blood flow changes and the ACh level and choline-acetyltransferase (ChAT), an enzyme that catalyzes ACh synthesis, using the same research model.\n\n\nData availability\n\nDataset 1: Raw data for cytoglobin, neuroglobin and acetylcholinesterase specific activity measurements 10.5256/f1000research.13592.d19700619", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by grants from Hibah PITTA 2006-DRPM Universitas Indonesia and supported by Indonesian Air Force Institute, LAKESPRA dr. Saryanto, Jakarta.\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\nWardaya and his team (from Aerophysiology Department of Aindonesian Air Force Institute, LAKESPRA dr. Saryanto, Jakarta) for helping the induction of hypobaric hypoxia, Ondi Sutisna and Arif Nurdianto (from Molecular Laboratory for Oxidative Stress Studies, Department of Biochemistry & Molecular Biology, Faculty of Medicine Universitas Indonesia) for some reagents preparation, and dr. Shanty Olivia Febriyanti Jasirwan, Sp.OG from the Writing Center FKUI helping proofread this article.\n\n\nReferences\n\nTosqui P, Colombo MF: Neuroglobin and cytoglobin: Two new members of globin family. Rev Bras Hematol Hemoter. 2011; 33(4): 307–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMudjihartini N, Nurhayati L, Saekhu M, et al.: Responses of brain tissues against the hypoxic condition in hemorrhagic stroke patients: Neuroglobin expression in brain tissue and plasma. AJPCR. 2017; 10(2): 407–9. Reference Source\n\nRatnayani: Cytoglobin Expression and Its Relation to Oxidative Stress in Blood and Brain Tissue of Hemorrhagic Stroke Patients. Master Thesis (unpublished). Master Program in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia. Jakarta. 2013.\n\nMudjihartini N: The expression pattern of neuroglobin and cytoglobin in the rat brain tissues and its correlation with the apoptosis as molecular adaptive response to the chronic systemic hypoxic condition. Doctoral Thesis (unpublished). Doctoral Program in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia. Jakarta. 2015. Reference Source\n\nKandel ER, Schwartz JH, Jessel TM, et al.: Principles of neural science. 5th ed. USA The McGraw-Hill Companies, Inc; 2013. Reference Source\n\nAndriani A, Prijanti AR, Mudjihartini N, et al.: Systemic Hypoxia Effect on Rat Brain Malondialdehyde, Glial Fibrillary Acidic Protein, and Acetylcholine Esterase Activity. eJKI. 2016; 4(2): 112–8. Publisher Full Text\n\nNi JW, Matsumoto K, Li HB, et al.: Neuronal damage and decrease of central acetylcholine level following permanent occlusion of bilateral common carotid arteries in rat. Brain Res. 1995; 673(2): 290–6. PubMed Abstract \| Publisher Full Text\n\nMulyawan W: Brain tissue adaptation response after intermittent hypobaric hypoxia induction in rat: Specific assesment toward Hypoxia inducible factor-1α. Doctoral Thesis (unpublished). Doctoral Program in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia. Jakarta. 2012.\n\nFago A, Hundahl C, Malte H, et al.: Functional properties of neuroglobin and cytoglobin. Insights into the ancestral physiological roles of globins. IUBMB Life. 2004; 56(11–12): 689–96. PubMed Abstract \| Publisher Full Text\n\nBurmester T, Haberkamp M, Mitz S, et al.: Neuroglobin and cytoglobin: genes, proteins and evolution. IUBMB Life. 2004; 56(11–12): 703–7. PubMed Abstract \| Publisher Full Text\n\nMuthuraju S, Maiti P, Pati S, et al.: Role of cholinergic markers on memory function of rats exposed to hypobaric hypoxia. Eur J Pharmacol. 2011; 672(1–3): 96–105. PubMed Abstract \| Publisher Full Text\n\nBohnen NI, Kaufer DI, Hendrickson R, et al.: Cognitive correlates of alterations in acetylcholinesterase in Alzheimer’s disease. Neurosci Lett. 2005; 380(1–2): 127–32. PubMed Abstract \| Publisher Full Text\n\nKellogg DL Jr, Zhao JL, Coey U, et al.: Acetylcholine-induced vasodilation is mediated by nitric oxide and prostaglandins in human skin. J Appl Physiol (1985). 2005; 98(2): 629–632. PubMed Abstract \| Publisher Full Text\n\nTong XK, Hamel E: Regional cholinergic denervation of cortical microvessels and nitric oxide synthase-containing neurons in Alzheimer’s disease. Neuroscience. 1999; 92(1): 163–75. PubMed Abstract \| Publisher Full Text\n\nZlokovic BV: Neurovascular mechanisms of Alzheimer’s neurodegeneration. Trends Neurosci. 2005; 28(4): 202–8. In: Van Beek AHEA, Claassen JAHR. The cerebrovascular role of the cholinergic neural system in Alzheimer’s disease. Behav brain Res. 2011; 221: 537–42. PubMed Abstract \| Publisher Full Text\n\nRosengarten B, Paulsen S, Burr O, et al.: Neurovascular coupling in Alzheimer patients: Effect of acetylcholine-esterase inhibitors. Neurobiol Aging. 2009; 30(12): 1918–23. PubMed Abstract \| Publisher Full Text\n\nSingh M, Kaur M, Kukreja H, et al.: Acetylcholinesterase inhibitors as Alzheimer therapy: From nerve toxins to neuroprotection. Eur J Med Chem. 2013; 70: 165–88. PubMed Abstract \| Publisher Full Text\n\nFarhan FS: Neuroplasticity as a response of physiological adaptation after intermittent hypobaric hypoxia induction in Sprague-Dawley rats. Doctoral Thesis (unpublished). Doctoral Program in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia. Jakarta. 2016.\n\nMasengi ASR, Farhan FS, Mulyawan W, et al.: Dataset 1 in: Cytoglobin, Neuroglobin, and Acetylcholinesterase Activity in Rat Brain as Adaptation Responses to Intermittent Hypobaric Hypoxia. F1000Research. 2018. Data Source" }	[ { "id": "32761", "date": "23 Apr 2018", "name": "Elena Rybnikova", "expertise": [ "Reviewer Expertise Hypoxia/ischemia", "hypobaric hypoxia", "brain", "neuroprotection", "cerebral mechanisms of hypoxic tolerance", "expression of genes and proteins", "neurochemistry" ], "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 paper by Masengi et al. reports that periodic hypobaric hypoxia up-regulates cytoglobin and neuroglobin levels and affects AChE activity in the brain. The study is potentially interesting but needs extensive revision to reach the level of acceptance.\nMajor criticism:\nThe model used cannot be referred to as an “intermittent (hypobaric) hypoxia”. In fact this is periodic (or repetitive) hypoxia. Intermittent hypoxia is a mode when lasting hypoxia is interrupted by brief episodes of normoxia, and this phenomenon is widely studied worldwide.\n\nThe mode of ascending the altitude looks puzzling: very fast elevation to a height about 10 km (speed is about 1.5 km/min that can itself produce many side-effects), and strange dynamics of the whole procedure. The feasibility of such model must be proved and explained to the reader. The authors refer to unpublished PhD thesis of Mulyawan (2012), as well as to the poster at The 1st Asian Researcher Symposium at the Universitas Indonesia-Depok, that cannot be accepted as reliable justification of the model.\n\nNeither the negative effect of single exposure nor the protective action of the repetitive exposures was demonstrated or even convincingly cited. The only reference to unpublished PhD thesis of Farhan (2016) reporting some cognitive improvement is not enough.\n\nInterpretation of the results needs much attention, e.g. no statistic differences with control are seen following AHH for Cygb and Ngb but authors write: \"In this research, Cygb and Ngb expressions of AHH group were decreased compared to normoxia\". Similarly, no differences in Cygb and Ngb expression where detected between IHH2x and IHH3x, therefore it cannot be interpreted as “especially IHH3x”, etc.\n\nMethods section, page 3, “After treatment…” - It is unclear at what time after hypoxic exposures the tissue samples were obtained. In case if the samples were taken immediately after hypoxia (that is 1 h from the onset of hypoxic trial) it is possible that it is not enough time to see the changes at the level of proteins. This might account for no changes revealed after single HH episodes (termed AHH). In this case it is a methodological failure.\n\nMinor questions:\nFig. 5 is redundant; it litters the article and should be removed. It adds no information.\n\nSome concern arises regarding the data on AChE activity and their interpretation. It is incorrect to conclude on the changes in cholinergic system based only on the parameter examined. We would recommend to additionally examine the AChE localization and its isoform composition. A rate of synthesis (ChAT activity) vs. degradation (AChE activity) processes should be also kept in mind. In addition, AChE performs actions other than degradation of Ach, e.g. is involved in cellular adhesion processes which play roles in neuroplasticity and neuroprotection.\n\nThe language style and grammar should be thoroughly edited.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "35986", "date": "19 Jul 2018", "name": "Joan Ramon Torrella", "expertise": [ "Reviewer Expertise Intermittent hypobaric hipoxia", "exercisephysiology", "altitude acclimatization" ], "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\nSummary The article studies the effects of hypobaric hypoxia bouts interspersed by 7 days on the levels of cytoglobin, neuroglobin and AChE activity in the brain of the rat. Although some interesting results are reported, we think it is not indexable in its present form.\n\nIntroduction\nThe principal concepts related to the investigation that is aimed to be developed in this article must be better addressed. For example, what is expected to happen with levels of Cygb and Hgb in the brain after acute hypoxia exposure? Why? [Are the references provided regarding to stroke a valid example of hypoxia? Should not be anoxia instead?] The same applies to AChE activity. Which is the physiological meaning of increasing AChE after chronic normobaric hypoxia exposure in reducing the cholinergic system function? In the reference provided on bilateral common carotid occlusion, is there any implication of increased AChE activity in reducing ACh levels in the hippocampus? Apart from reference 4 (which is an unpublished study), are there any other references relating HIF1α levels with Cybg and Hgb levels and/or AChE activity? In our opinion, this is an important consideration to include in the Introduction section since it is at the basis of the authors’ hypothesis and the aim of the study. Provide additional references to support that HIF-1α is increased in rat brain after exposure to IHH, since reference 8 is a non-published work.\nMethods\nA subheading entitled “Hypobaric hypoxia procedure” should be included after the fourth paragraph. We have some concerns on the intermittent protocol used. In our opinion, an intermittent protocol is applied when the individuals are submitted regularly, during short periods of time, to a stimulus (hypoxia in this case). In the present protocol, rats were submitted only three times to the hypoxic procedure and interspersed by a long period (7 days). We have serious concerns to admit that this could be considered “intermittent” hypoxia. What kind of exposure (or risk) this protocol try to mimic? The reader needs some explanation about why the authors chose this particular exposure model. Although probably it will not alter the results of the experiment, chronical exposure of experimental animals to shredded newspaper is not desirable and must be avoided because the potential detrimental effects on their health caused by the inhalation of paper dust or direct nose and mouth contact with newspaper ink. Statistical analysis. Please, indicate the dispersion parameter used throughout the figures: standard deviation (SD)? Variance? Standard error of the mean (SEM)? Coefficient of variation (CV)?\nResults\nProvide reference for Federer’s rule and explain what does this rule has to do with the groups considered for the experiment. A paragraph indicating the usefulness and applicability of the hypoxia exposure protocol performed in this work is needed. Which is the rationale of applying the “hypobaric hypoxia type I chamber flight profile”? For what purposes was designed this protocol and what will be the translation of the hypothetic results obtained? Figure-5c. Which units are represented in Y axis? Why there is not dispersion parameter in the bar diagrams? In general, whisker plots or boxplots can replace histograms in Figures 2, 3, 4 and 5.\nDiscussion\nFigure 5 is not discussed in this section. Authors must include some comments on that Figure justifying the relevance of the data presented and including some comments on the information provided by this Figure. The fourth paragraph of the Discussion section would be a good place to do that. The first paragraph must clearly explain, using references, the mechanisms why Cygb and Ngb levels are decreased after AHH exposure. Additionally, it is not clear enough the physiological mechanism, hypothetic or supported by previous literature, by which IHH induce increases in Cygb and Ngb that provide adaptive protection against hypoxia1-2. Relationship between HIF-1α levels triggered after hypoxia exposures and the studied proteins should be commented. We suggest also developing the possible implementation of this research results for individual training to adapt to hypoxia conditions (this is only mentioned in the two last lines of the Discussion). Which new insights introduce this study to, for example, pilot or parachute training? Which are the training protocols currently used and what would the results presented in this study add to this field?\nGeneral The final edition of the text must be reviewed since there are several grammatical and language mistakes.\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": [] } ]	1	https://f1000research.com/articles/7-426
https://f1000research.com/articles/7-425/v1	04 Apr 18	{ "type": "Research Article", "title": "Factors influencing the decision to commit violence in Thai male juvenile offenders: A phenomenological study", "authors": [ "Wasinee Wongin", "Suchada Paileeklee", "Wasinee Wongin" ], "abstract": "Background: Violence is a social problem that affects the physical and mental health of adolescents. For a long time, Thailand has adopted strategies formulated by the World Health Organization to reduce violence but has been unsuccessful. The aim of the current qualitative study was to understand the decision of adolescents to commit violence and to identify factors contributing to violence among male juvenile delinquents. Methods: Data were collected from 50 male juvenile offenders at the Department of Juvenile Observation and Protection detention facilities located in 5 regions of Thailand through in-depth interviews focusing on delinquent violence committed in the past year. Results: Adolescents who decide to use violence have been associated with and live in environments where they face conflicts in their neighborhood and violence in their community. Mostly, juveniles were found to drop out of school, engage in abuse and supply of drugs, consume alcohol, and experienced domestic violence problems and family divorce. Juvenile offenders typically experience and learn about violence from family and peers, which creates a positive attitude toward violent behavior in them. These offenses can be categorized into intentional violence, which involves seeking revenge or resolving prior conflicts and requires premeditation, and unintentional violence, which results from a situation escalating quickly and usually requiring no preplanning, such as insults, conflicts, power struggles, self-defense, or protecting peers. Conclusions: A violence prevention model and guidelines need to be introduced into Thailand’s youth health care system. This study identified a lack of both decision-making skills and socially adequate adjustment to difficult situations among adolescent perpetrators as precursors to violent behavior.", "keywords": [ "decision", "perpetration", "violence", "adolescents", "delinquency" ], "content": "Introduction\n\nViolent behavior and delinquency have become a major global public health issue (WHO, 2016). According to the World Health Organization (WHO), 43% of homicides are committed by juvenile offenders every year, and most occur among youth aged 10–19 (WHO, 2016). Fighting and bullying are evidently common among adolescents. The rate of adolescent homicides saw a greater decrease in high-income countries than in low- and middle-income countries during the 2000–2012 period (WHO, 2016). Guns are the number one cause of violence and death in juvenile homicide cases (Gudlaugsdottir et al., 2004; Reingle et al., 2013; WHO, 2016). For each adolescent killed, many more sustain injuries (WHO, 2010) that often require hospital treatment. Firearm attacks end more often in fatal injuries than assaults involving fists, feet, knives, and blunt objects. Adolescent violence greatly increases the costs of health, welfare, and criminal justice services; reduces productivity; and decreases the value of property (WHO, 2016).\n\nAdolescents may experience violence at any time and place. Causes of violence are deeply rooted in pre-established social and cultural structures of human existence. Contributing factors to violence include individual factors prescribing aggressive behaviors such as family relations and conflicts (Herrenkohl et al., 2012), and other cultural or external factors that may induce violence (Krug et al., 2002). Several studies on adolescents suggest that the intention to use violence or violent behavior is associated with a number of individual, family, peer, gang, and contextual factors (Gudlaugsdottir et al., 2004; Henry, 2012). Almost all studies agree that males are more likely to commit violence than females (Gudlaugsdottir et al., 2004; Henry, 2012). This is because females have better capacity for stress management and solve problems in situations that are less likely to cause a serious incident than males (Bennett et al., 2005). Some studies report rates of crime against age; a sharp increase in criminal activity in mid-adolescence followed by an equally sharp decline in these rates in early adulthood have been noted (Blonigen, 2010).\n\nAdolescents who have experienced more than four negative life events or exposure to violence in the past year and currently engage in substance and alcohol abuse are more likely to commit violence than adolescents with no negative life events or exposure to violence and no engagement in substance and alcohol abuse (Gorman-Smith & Tolan, 1998; Gudlaugsdottir et al., 2004; Henry, 2012; Loeber et al., 1998). Adolescent gang membership and associating with delinquent peers increases the likelihood that at-risk adolescents will engage in violent behavior and delinquency (Decker et al., 2013). The more risk factors for violence an individual possesses, the higher his or her likelihood is of joining a gang (Melde et al., 2012). Many studies indicate a number of risk factors including a history of prior violence, early onset of violent behavior, high levels of early aggression, low guilt, positive violent attitudes, negative school environments, and dropping out from school (Gorman-Smith & Tolan, 1998; Loeber et al., 1998). A cluster of risk factors in adolescents often explains the cause of violence, such as cases of sexually abused children that can commonly be traced back to poverty-stricken families in overcrowded inner-city neighborhoods in which they receive little attention from their parents (Herrenkohl et al., 2012; Krug et al., 2002).\n\nThe purpose of this study was to understand the phenomena underlying the violence committed by juvenile delinquents, who have a severe problem of violent behavior. Many studies have identified risk and protective factors through quantitative approaches in an attempt to explain adolescent violence. However, only a few studies explain violent behaviors in adolescents through qualitative analysis. In addition, other studies have examined the relationship among factors influencing violent behaviors and proposed interventions that have attempted to reduce the highest risk factors but have actually been unsuccessful. Finally, the issue of adolescent violent crime is significantly more severe and complex than other delinquencies, thus requiring a qualitative study that can accumulate key knowledge to better understand the decision made by adolescents.\n\nThis qualitative study uses a grounded theory approach to describe the phenomena of serious adolescent violence among male juvenile offenders, whose violence, which results in severe injury or death of their victims, has a considerably more severe impact than that of general adolescents. This study focuses on adolescents’ decision to use violence in conflict and difficult situations and emphasizes that the influencing factors are multi-faceted. The findings can inform the design of appropriate prevention programs that address factors contributing to violent behavior in adolescence.\n\n\nMethods\n\nData were collected from December 2014 to April 2015 through in-depth interviews (Supplementary File 1). The questions asked about 1) general information, including age, education, occupation, family status, behavioral description, type of perpetration, and drug use; 2) method of violence; and 3) purposes and elements of the decision-making process. The questions were designed to allow key participants to provide significant information aligned with the study concept.\n\nA total of 50 juvenile offenders were recruited from the Department of Juvenile Observation and Protection detention facilities located in five regions of Thailand. At any given time, each facility provides services to approximately 80 adolescent offenders, all of whom were sentenced by the court. The majority of the arrests involved intent to assault, carrying weapons with intent to harm, possession of firearms, sexual assault, and murder in some cases. Purposive sampling was used to select participants aged between 15 and 19 who had a history of violence and arrests (excluding individuals who had been indicted and released or sent to other institutions by the court) and were detained at one of the five Department of Juvenile Observation and Protection detention facilities.\n\nAll participants were informed about the voluntary and confidential nature of their participation in the study. All the information collected for the study was confidential; hence, it was not shared with the detention officers or permitted to affect the ongoing trial of the participants. Approval for the study was obtained from the Research Ethics Committee of Khon Kaen University (approval number HE571317). The Research Ethics Committee waived the requisition of written consent from participants and their parents, due to illiteracy and a high frequency of changing residence. Prior to interviewing, informed verbal consent was sought from the participants’ legal guardians (as approved by the Research Ethics Committee). Only the researcher was present during the interviews. The researcher began by introducing themselves, stating the purpose of the interview and seeking verbal consent to continue from the interviewee. The interviews were conducted in a private room and lasted 45–60 minutes per participant. The interviews were recorded, and notes were taken by the researcher. The recordings and transcript files were destroyed after the data analysis was completed.\n\nStep 1: Preparation for the interview. The researcher set up appointment dates and times to collect data from each target facility.\n\nStep 2: Introduction to the interview. The researcher established a rapport with the participant by initiating conversation about living conditions and adjustment to the facilities. This strategy aimed to build trust and create a friendly vibe during the interview. The researcher then sought information about the participants’ history of violence, regardless of whether they were indicted. The researcher listened and sincerely acknowledged what the participants had to say while they shared their stories without imposing any opinion, suggestions, or judgment.\n\nStep 3: The interview. The researcher began with the following important questions: “What crime brought you here?,” “Can you tell me what happened during that crime?,” “What is the most violent crime or incident you have inflicted on others? When, where, and how did it happen?,” “Were you or any other persons affected by that crime/incident?,” and “Why did you decide to use violence at that time?” The researcher’s duty was to acknowledge the participant during the interview while also closely observing facial expressions. The questions needed to be repeated or revisited at times in order to allow the participants to provide further details so that the researcher could obtain enough information to cover all the three study concepts (general information, method of violence, and purposes and elements of the decision-making process). The interview adopted a naturalistic inquiry style; therefore, there was no particular order to questioning, although note taking had to conducted throughout for mapping the thinking process following transcription after completing each individual encounter. The participants were free to inquire about any acquired information at any point during the interview. In addition, the researcher disclosed the notes taken to the participants at the end of the interview.\n\nStep 4: Conclusion of the interview. The interviews were concluded in case of the following: 1) the researcher felt that sufficient data had been collected; and 2) the researcher noted uneasiness, lack of interest, or constraint in the participant. The researcher used a tactic to conclude the interview by requesting the participant to sum up the main points, thoughts, and experiences that had been shared during the interview in order to prevent any misinterpretation. The process of interview conclusion also included reaffirmation of confidentiality, privacy of information collected and the fact that the provided information would not affect the participant’s ongoing trial.\n\nStep 5: Data analysis, generating categories, and creating hypotheses from data. Once data were collected from the participants, the researcher used data coding to categorize information. The coding was organized into 1) acts: violent behaviors; 2) participation: the participant’s involvement in the incident, namely instigator or providing reinforcement to help friends; 3) relationship: status of relationship with those involved in the violence, such as close friend, lover, or relatives; and 4) setting: the violent crime context and the living environment context.\n\nOnce data were collected from the participants, the researcher used data coding to categorize information. Data analysis was conducted by considering the essence of the content and using the description to describe the phenomenon of adolescent violence (Russell, 1984).\n\n\nResults\n\nThe sample group was composed of all males with an average age of 16.58 years. All the selected adolescents were indicted with charges related to inflicting harm on others (assault, attempted murder, murder, and violation of the Firearms Act). Eight in ten adolescents came from broken homes. Half of the group had divorced or separated parents, and one or both parents were deceased. Half of the group also was raised by grandparents, or a parent and a step-parent (21%). Moreover, 28.8% of the adolescents lived independently without supervision. Reasons for broken homes were parents suffering from alcohol addiction, gambling, violent quarrels, financial disputes, or imprisonment of parents. Among those raised by grandparents or relatives, some were spoiled, some were brought up without any structure, and some were treated contemptuously by relatives. Without structure, adolescents were free to hang out with friends or at game shops, crawl pubs or join motorcycle gangs, begin drinking and using drugs, and enter gang fighting. Group acceptance and friendship guaranteed self-importance, safety, and stability, which compensated for the lack of love or the feeling of being unwanted that emerged from the separation of their parents. Those who lived without supervision were either alone or with friends or lovers. Life without supervision implied making decisions and managing problems without guidance from adults.\n\nMost participants decided on their own to drop out of school in the middle of a school year at middle or high school. Half of the participants left school owing to history of truancy, financial hardship, or indictment and detention, which resulted in absenteeism and missing exams. In total, 80% used illegal substances, with some starting as young as the age of eight. The most popular drugs were methamphetamine, cannabis, Kratom (Mitragynaspeciosa), inhalants, and Alprazolam. Most adolescents used more than one drug.\n\n“I had to drop out of school because I had a fight with my friends. My mother was invited to meet the student welfare teacher because I got into fights twice a month.” (Northern Thailand)\n\n“I always skipped classes to smoke with friends behind the toilet wall. I did not attend classes or do homework, so I failed all my exams.” (North Eastern Thailand)\n\n“I always go to the back of the school toilet to smoke with friends. I do not attend classes and never complete my homework; I miss all exams.” (North Eastern Thailand)\n\nMost adolescents began using violence by engaging in bullying in their school or community. Nine out of ten participants were bullied and hence responded with violence or bullied others in the same school. School gangs easily clash, and this often escalated to violent confrontations. Some adolescents picked fights with their teachers, blaming them for pressuring or bullying them. Such bullying included using degrading language, such as insulting parents, teasing students to make them feel inferior, or more severe punishments such as throwing blackboard erasers at students. School bullying led to violence among adolescents. Half of the adolescents in this study reported being expelled in the middle of the school year because of frequent violent confrontations with friends, classmates, or teachers. At times, the violence involved getting into a brawl with the children of school administrators, or the disputants were severely injured and were admitted to hospital. These incidents forced some delinquents to never return to school. Without school and excessive time on their hands, some adolescents found work while others joined gangs with other delinquents. This acted as an entry point to a world full of risks of violence and other law violations.\n\nIn addition to bullying in a school, it was found that bullying outside of school also led to violence. Ten percent of the sample group escalated disputes by insulting and threatening with weapons such as spray shooting or throwing bottles while driving by on a motor bike near the homes of their rivals in order to create fear. Moreover, bullying through social media involved adolescents posting pictures of firearms, knives, and a message to challenge their rivals in order to show authority and threaten them to create fear.\n\n“I had a physical fight with my friend at school for the first time after physical education class.” (Southern Thailand)\n\n“My first fight was when I was 13 years old. I went with my brother next door. We were very close and often hung out together.” (Northern Thailand)\n\n“My fights began when I was a high school student. There was a senior from another school threatening to hurt my friend, and I was called to help. We fought. Guns were fired. And we became arch enemies since then…” (Southern Thailand)\n\nIn conflict situations, adolescents need to decide how to respond. The decision depends on the situation and elements, both of which influence the choice to use or not use interpersonal violence.\n\n“I had a fight with a teenager in the pub. After he left, I and my brother followed him and used a knife to stab him 3–4 times through his heart until he died.” (Southern Thailand)\n\n“A teenager from other regions came to the local event. He teased my girlfriend. I got very angry, so I shot him after the event was finished” (Southern Thailand)\n\nAdolescent decisions were divided into two scenarios: intentionally violent scenarios and unintentionally violent scenarios (Figure 1). In intentionally violent situations, adolescent perpetrators aimed to seek revenge, terminate the enemy, and resolve problems. Violence was justified as a means to an end for conflicts between gangs, villages, and enemies or rivals. The premeditation included a recruitment process, such as using social media; gaining more supporters; including a female bait to lure the rivals to a meeting point at times; and locating weapons, such as bombs, knives, and firearms. Each incident would be executed differently; for example, a bomb would be thrown to disperse the target group followed by stabbing and shooting, or a duel using guns or knives with the purpose to kill would be arranged.\n\nIn unintentionally violent situations or unplanned violence, the objective of using violence was often to show authority or self-defense in response to a threat, assist or rescue friends to prove gang spirit, insult, or respond to gang territory crossing. Victims were individuals who crossed gang territory or simply some strangers. Weapons used for self-defense included brass knuckles and pocket knives; guns; or anything that could be found, such as sticks, beer bottles, or other materials. The method of violence was fist fighting without weapons or using any item to inflict injury, punching with brass brackets, stabbing with knives, and shooting with guns.\n\nFour out of ten adolescents committed violence in the streets, such as while riding motorbikes past rival gangs or crossing gang territories. The fighting involved knife fighting, gun shooting, use of sticks to assault, and motorcycle trapping. These street fights caused serious injuries resulting in admittance to the hospitals and death of two victims.\n\nElements of the decision to use violence involved other people, such as family members, relatives, peers, and lovers, owing to their close relationship with the adolescent. Those who had experienced violence were more likely to commit violence. Alcohol consumption and drug use were found to be among the key variables triggering adolescent violence. A large number of adolescent offenders committed violent crimes under the influence of substances and alcohol. Six in ten adolescent perpetrators spoke about easy access to weapons, and their own weapons, such as guns, Spatha or Samurai knives, brass brackets, and bombs. Adolescents who hung out as a group and had rivals or enemies always carried weapons. They obtained these weapons by purchasing these on their own, from friends, making them themselves, trading with drugs, or borrowing. Weapons such as brass brackets, Spatha or Samurai knives, and pocket knives were affordable and easy to find in common marketplaces. Some adolescents produced homemade weapons such as bombs, homemade pen guns and cap guns, and Spatha or Samurai knives by using skills learned from friends or techniques from lessons taught at schools or jobs. In addition, adolescents knew the sources of distribution and purchasing processes of guns.\n\n“There were fights almost every day. Before every fight, I would take an Amphetamine tablet. If I did not take them, I would not dare to do it—pulling the trigger.” (Southern Thailand)\n\n“Every day, before going out for some entertainment, I would take two tablets of Alprazolam. I have stabbed people three times during night-outs. I have never waited to see if they died or not. I got very upset at my brother for warning me about taking Alprazolam before going out, so I tried to stab him with the knife too.” (Southern Thailand)\n\nApart from narcotics, the study found that alcohol is also another crucial variable dictating violent incidents among juvenile offenders. Many juvenile offenders have committed crimes related to violence while being under the influence of alcoholic beverages, which are spirits and traditional herbal liquor. These groups of juvenile offenders consume alcoholic beverages with friends and later come out to fight and use violence, as the following examples indicate:\n\n“Every day after work, I would go out to drink alcohol or beer. After I got drunk, I would dance in front of the stage and fight with others…” (Eastern Thailand)\n\n“That night, I was drinking and got drunk in a shop in my village. A guy came in the shop to buy things. I borrowed his motorbike, but he did not want to give [it to] me. I got angry, so I hit his head with a stone.” (Northern Thailand)\n\nOther people, such as family members, peers, gangs, cousins, and lovers, influenced adolescents’ decision to use violence in both types of scenarios. Seven out of ten adolescents joined their peers and adolescents in their neighborhood to form gangs of four members or more. Half of them were in a gang with 4–10 members. 1 in 3 was in a gang with 11–20 members. 3 in 10 adolescents belonged to gangs with 30 or more members. Large-sized gangs engaged in selling illegal substances and motorcycle races. Most gang activities included drinking alcohol, nightclub crawling, drug abuse, and speed motorcycle racing. Adolescents deemed these activities as normal and in trend. Moreover, the activities they did together signified belonging, bravery, and an exhibition of talents. Most importantly, there was a transfer of violent culture. Gang violence was often triggered by cross-territory insults that led to conflict and fighting.\n\n“Our group has about ten people. We love each other a lot. Nobody can mess with my friends. All my criminal cases are because of these friends.” (Southern Thailand)\n\n“The closest person to me is my big brother. I love him a lot and can do anything for him. Nobody is allowed to hurt him because we only have each other.” (Southern Thailand)\n\n“The beginning of my fight was because of one of my close brothers who had an arch enemy. He took me to my first fight. I was told that if I refused to fight, I would definitely get hit by his brass knuckles. So I had no choice.” (Southern Thailand)\n\nAdolescents exhibiting violent behavior were found to live in high risk or vulnerable communities, such as communities with drug addicts and those near pubs, bars, snooker tables, karaoke bars, or gambling houses. These locations inevitably became meeting points or killing spots for gangs. Communities and youth living environments play an important role in making illegal behavior decisions and lead to violent decisions or conflicts with others.\n\nMost juvenile offenders were stigmatized as delinquents and trouble makers. This indicates that community members also influence violence in juvenile offenders. If their group formations or deviant behaviors are being negatively judged by family or community members as trouble makers for the family or society, the stigma can put juvenile offenders under the pressure. This leads to vulnerability about peer pressure, and because peers provide them with confidence, they behave in a manner that takes both the pressure ad stigma into account.\n\n“The villagers look at me as a bad boy, an inhalant addict or a night partier.” (North Eastern Region)\n\n\nDiscussion\n\nThe phenomenon of adolescent violence by juvenile delinquents is complex and multi-faceted. Unlike other delinquencies, violent adolescents often escalate the level of disputes and degree of impact on the victim as well as the instigator. This is because violence is deemed as the most effective conflict resolution, which is also justified by the personal desire to seek violence; the belief that violence is a normal adolescent behavior; outcomes that can be measured, such as the injuries to the enemy or surrenders; and the conceivable power within the so-called territory.\n\nViolence-seeking behavior in adolescents often originates from bullying in school (Olweus, 2011; Renda et al., 2011). This evidence highlights poor bullying prevention and management in school (Monks et al., 2009). Adolescents who are involved in bullying, either as a recipient or a perpetrator, often respond to provocations with violence, which then escalates to gang violence. In gangs, adolescents undergo initiation processes, which are designed to modify behaviors. Gang culture instills a belief system and normalizes violence and delinquency (Alleyne & Wood, 2010). The nature of violence eventually leads to criminal intent, including robbery, which involves premeditation (planning, identifying of victim) and fighting in public or expressive crime. This type of violence is an indication of a lack of adequate self-control.\n\nTransition into adolescence involves drastic changes in hormones and, consequently, temperament. Responses to situations can involve extreme emotions or violence and lack of self-control. The decision to resort to violence is purposive (Monks et al., 2009) depending on the situation. Additional influences include peer pressure, family relations, neighbors, and community (Sherer & Sherer, 2011), and drug and alcohol use often fuels the intensity and decisiveness in committing violence (Baskin–Sommers & Sommers, 2006; Fagan et al., 2015; Gudlaugsdottir et al., 2004; Swamhn & Donnovan, 2004; Tyner & Fremouw, 2008). Moreover, adolescent offenders usually find it easier to access weapons than their peers (Bingenheimer et al., 2005)\n\nAdolescents undergo transformation of physicality, mind, emotional maturity, and adjust to society, peers, and school as they transition into adulthood. It is important to understand that adolescents are naturally curious to try new things and often seek challenges. If violence is embedded in their environment – family, peers, schools, community, society – adolescents would likely fall in the path of violence and delinquency with no regard to the consequences. Delinquent adolescents acquire different life skills than their law-abiding counterparts. Using violence to solve problems is a common value of the group, but is also related to other risk factors and use of illegal substances.\n\nThe findings of this study suggest that intervention programs should be offered to aid adolescent offenders during probation with emphasis to prevent repeat offenses, which could escalate to higher-level crimes in the future. Interventions must target and offer help to those who are developing life skills, such as decision-making skills, rejection skills, and problem-solving skills. This population lacks the opportunity to refine these skills, as they might have been neglected at a young age or due to childhood trauma and school dropouts. Environmental management is highly influential in these behaviors. Appropriate management and control of liquor stores, gambling places, games shops, and internet shops, as well as effective weapon control and limiting of weapon access to adolescent offenders should be implemented. Strict and clear policies and regulations will reduce risk factors, thereby creating community safety.\n\nThe present sample included adolescent offenders who were detained at Department of Juvenile Observation and Protection detention facilities; therefore, they are not representative of all teens. In addition, the researcher could not evaluate during interviews other confounders such as mental health problems.\n\n\nConclusions\n\nThis study aimed to understand juvenile offenders’ decision to use violence in problem or conflict situations. Male juvenile offenders who had engaged in serious violence were chosen for the study. It was found that violent behaviors in male juvenile offenders originate from bullies in schools or communities, leading to behaviors of fighting or using violence in order to solve conflicts that occur among juvenile groups. The level of violence gradually escalates to serious violence, resulting in injuries or death in violent flights. Two types of decisions considered from various elements are made by male juvenile offenders when using violence, intentional use of violence and unintentional use of violence. However, the principal variables affecting both situations of decision in using violence are close friends and gangs, which eliminate their hesitancy to attack others in order to protect their friends, and the dignity of their group or gang. As the majority of juvenile offenders are from broken families, they consider their relationships within their groups of friends as something to be proud of, in order to replace the lack of love or respect received from families. It was found that most of the misbehaviors were engaged in under influences of narcotics and alcohol. Furthermore, the study found that these juvenile offenders possess the ability to produce deadly weapons such as guns, grenades, and sword-knives, and can immediately procure them if needed. Another interesting aspect is that these groups of juvenile offenders utilize fighting as a solution to manage issues between cliques or persons.\n\nLastly, male juvenile offenders were found to lack decision-making skills as well as social adequacy. Therefore, creating a violence prevention model and formulating guidelines are crucial and should be assigned as part of juvenile health care systems. Additionally, studies regarding prevention that can resolve violence in juvenile offenders are required.\n\n\nData availability\n\nRecordings and transcript files are not available. To maintain participant confidentiality, these files were destroyed immediately following data analysis. Themes and quotes from the data analysis are available in Thai. This data can be obtained on approval from the Ethical Committee of Khon Kean University. To apply, please visit the Ethical Committee of Khon Kean University webpage (https://eckku.kku.ac.th) or contact the corresponding author.\n\n\nConsent\n\nThe Research Ethics Committee waived the requisition of consent from participants. Prior to interviewing, informed verbal consent was sought from the participants’ legal guardians.", "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 the Department of Juvenile Observation and Protection, Thailand.\n\n\nSupplementary material\n\nSupplementary File 1: Interview guide used in Thai with English translation.\n\nClick here to access the data.\n\n\nReferences\n\nAlleyne E, Wood JL: Gang involvement: Psychological and behavioral characteristics of gang members, peripheral youth, and nongang youth. Aggress Behav. 2010; 36(6): 423–436. PubMed Abstract \| Publisher Full Text\n\nBaskin-Sommers A, Sommers I: Methamphetamine use and violence among young adults. J Crim Justice. 2006; 34(6): 661–74. Publisher Full Text\n\nBennett S, Farington DP, Huesmann LR: Explaining gender differences in crime and violence: The importance of social cognitive skills. Aggress Violent Behav. 2005; 10(3): 263–288. Publisher Full Text\n\nBingenheimer JB, Brennan RT, Earls FJ: Firearm violence exposure and serious violent behavior. Science. 2005; 308(5726): 1323–1326. PubMed Abstract \| Publisher Full Text\n\nBlonigen DM: Explaining the relationship between age and crime: contributions from the developmental literature on personality. Clin Psychol Rev. 2010; 30(1): 89–100. PubMed Abstract \| Publisher Full Text\n\nDecker HS, Melde C, Pyrooz DC: What do we know about gangs and gang members and where do we go from here? Journal justice quarterly. 2013; 30(3): 369–402. Publisher Full Text\n\nFagan AA, Wright EM, Pinchevsky GM: Exposure to violence, substance use, and neighborhood context. Soc Sci Res. 2015; 49: 314–326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGorman-Smith D, Tolan P: The role of exposure to community violence and developmental problems among inner-city youth. Dev Psychopathol. 1998; 10(1): 101–116. PubMed Abstract \| Publisher Full Text\n\nGudlaugsdottir GR, Vilhjalmsson R, Kristjansdottir G, et al.: Violent behaviour among adolescents in Iceland: a national survey. Int J Epidemiol. 2004; 33(5): 1046–1051. PubMed Abstract \| Publisher Full Text\n\nHenry DB: Mediators of effects of a selective family-focused violence prevention approach for middle school students. Prev Sci. 2012; 13(1): 1–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHerrenkohl TI, Lee J, Hawkins JD: Risk versus direct protective factors and youth violence: Seattle social development project. Am J Prev Med. 2012; 43(2 Suppl 1): S41–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrug EG, Dahlberg LL, Mercy JA: World report on violence and health. Geneva: World Health Organization. 2002. Reference Source\n\nLoeber R, Farrington DP, Waschbusch DA: Serious and violent juvenile offenders. In Loeber R, Farrington DP, editors. (Eds.), Serious and violent juvenile offenders: Risk factors and successful interventions. Thousand Oaks, CA: Sage. 1998; 13–29. Reference Source\n\nMelde C, Diem C, Drake G: Identifying correlates of stable gang membership. J Contemp Crim Justice. 2012; 28(4): 482–498. Publisher Full Text\n\nMonks CP, Smith PK, Naylor P, et al.: Bullying in different contexts: Commonalities, differences and the role of theory. Aggress Violent Behav. 2009; 14(2): 146–156. Publisher Full Text\n\nOlweus D: Bullying at school and later criminality: findings from three Swedish community samples of males. Crim Behav Ment Health. 2011; 21(2): 151–156. PubMed Abstract \| Publisher Full Text\n\nRenda J, Vassallo S, Edward B: Bullying in early adolescence and its association with anti-social behaviour, criminality and violence 6 and 10 years later. Crim Behav Ment Health. 2011; 21(2): 117–27. PubMed Abstract \| Publisher Full Text\n\nReingle JM, Jennings WG, Lynne-Landsman SD, et al.: Toward an understanding of risk and protective factors for violence among adolescent boys and men: A longitudinal analysis. J Adolesc Health. 2013; 52(4): 493–498. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRussell BH: Research methods in methods in anthropology. London New Delhi: Sage Publications. 1984.\n\nSherer PP, Sherer M: Violence among high school students in Thailand: CulturalPerspectives. Int J Intercult Relat. 2011; 35(6): 867–880. Publisher Full Text\n\nSwamhn MH, Donnovan JE: Correlates and predictors of violent behavior among adolescent drinkers. J Adolesc Health. 2004; 34(6): 480–492. PubMed Abstract \| Publisher Full Text\n\nTyner EA, Fremouw WJ: The relation of methamphetamine use and violence: A critical review. Aggress Violent Behav. 2008; 13(4): 285–297. Publisher Full Text\n\nWorld Health Organization: Violence prevention: the evidence. WHO Press, 2010. Reference Source\n\nWorld Health Organization: Youth violence. Fact sheet. Geneva: WHO Press, 2016. Reference Source" }	[ { "id": "34568", "date": "29 Jun 2018", "name": "Sang-arun Isaramalai", "expertise": [ "Reviewer Expertise Health promotion", "occupational health", "aged care", "community 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\n1. Incongruency on the philosophical basis of the research methodology between qualitative and quantitative has existed- using the term \"sample\" in results - page 4 and in Limitation in page 8 including terms, representative & confounders.\n2. Introduction- gap of knowledge was unclear-why need to explore those influencing factors, what have known and what need to be explored for resolving the problem.\n3. Using qualitative data analysis, grounded theory, themes are expected to be emerging from the data themselves not from known categories.\n\n4. The Procdures, page 4 need to take out the subject, the researcher.\n\n5. Figure 1, Need to include influencing factors in the diagram and provide discussion on how those factors mediate or moderate the decision.\n6. Discussion - page 7 need to explain why on the study findings not part of literature review.\n7. Discussion page 8 - study results from qualitative research are not ready for utilization or designing intervention.\n8. Conclusion - Not summary of the results, but need to focus on what was new knowledge emerging from the study, what confirmed existing knowledge.\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": "33977", "date": "31 Jul 2018", "name": "Marta Talavera", "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 theme is interesting and relevant, but the sample size is too small to be able to generalize results.\n\nAlso, it would be necessary to provide a better social and economic contexualization. The bibliography needs to be updated with more recent references. The methodological description is not clear. The exhibition is not detailed as well as the subsequent analysis, so the results do not have sufficient foundation for the statistics.\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? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-425
https://f1000research.com/articles/7-424/v1	04 Apr 18	{ "type": "Research Article", "title": "Prevalence of Autism Spectrum Disorder (ASD) among the children aged 18-36 months in a rural community of Bangladesh: A cross sectional study", "authors": [ "Shaheen Akhter", "A.H.M Enayet Hussain", "Jannatara Shefa", "Gopen Kumar Kundu", "Fazlur Rahman", "Animesh Biswas", "A.H.M Enayet Hussain", "Jannatara Shefa", "Gopen Kumar Kundu", "Fazlur Rahman", "Animesh Biswas" ], "abstract": "Background: Autism spectrum disorder (ASD) refers to a group of complex neurodevelopment disorders characterized by repetitive and characteristic patterns of behavior and difficulties with social communication and interaction. In Bangladesh, autism in children is a significant burden of disease. Early identification of ASD could improve quality of life. The study has explored at the prevalence of ASD among rural community children aged between 18-36 months. Methods: A cross sectional study was conducted among the 5286 children aged between 18-36 months in a rural community. Household level data was collected using screening tool MCHAT. Primarily screening positive 66 children were invited for final diagnosis in a health camp. Diagnosis was made by different staging started from primary screening, followed by validation using MCHAT and flash card. Final diagnosis was made by the paediatric neurologists, child clinical psychologists and development therapist using diagnostic tools (DSM-IV & ADOS). Results: 04 children were diagnosed with autism spectrum disorder (ASD). Prevalence of the ASD in rural community was found 0.75/1000 children. Among the four ASD cases three were boys and one was girl and age range was between 20- 30 months. Whereas, the highest prevalence rate found was for the cerebral palsy which was 5.6/1000 children and Developmental delay (2.6/1000) was the next to that. Conclusions: Age specific autism (18-36 months) in children is found higher in rural community of Bangladesh. In order to get more comprehensive information on autism in other age groups of children in rural community, further study is required. Early detection in rural community could help the policy makers to decentralization of health services among the ASD children in rural community.", "keywords": [ "Autism spectrum disorder (ASD)", "children", "prevalence", "rural community", "Bangladesh" ], "content": "Introduction\n\nIn recent years, epidemiological studies have shown a rapid increase in the prevalence of autism spectrum disorders (ASD)1,2. Throughout the world, it is reported to be 1 in 150 children (See Centre for Research and Information site). According to estimates of the Centre for Disease Control and Prevention (CDC)’s Autism and Developmental Disease Monitoring (ADDM) Network, approximately 1 in 68 children aged 8 years are identified with ASD3. California Department of Developmental Services (CDDS) and IDEA data sets are qualitatively consistent in suggesting a strong increase in autism prevalence over recent decades4. Prevalence studies from European countries, with an age range of birth to adulthood, varied from 1.9/10000 to 72/100005. A systematic review article reported differences in prevalence of autism in South Asia. It ranged from 0.09% in India to 1.07% in Sri Lanka6\n\nThe disease manifests at an early age in children, and is likely to last for life7. ASD severely affects the social functioning of an individual and may have a negative impact on the entire family of the affected individual1. The accuracy of the numbers regarding prevalence of ASD depends on diagnostic criteria, age and geographical location, service availability and awareness of ASD8. Advanced maternal and perinatal age is also a risk factor for ASD, with significantly increased risk with each 10-year increase in maternal age9.\n\nAverage prevalence of ASD in Asia was 1.9/10000 before 1980, while it is 14.8/10000 from 1980 to present10. The overall reported prevalence of ASD in recent studies was higher than previously reported in Asia.\n\nIn Bangladesh, it has been predicted that autism is an underestimated, yet significant health problem. In community studies done by Mullick and Rabbani in 2005 and 2009, autism was 0.2 and 0.84/1000 children respectively11,12. From a systematic review, the prevalence of ASD was found to be ranging from 0.15–0.8% in Bangladesh6. Cambridge medical university’s patient registration records showed an increased rate of autistic children seeking treatment, from 12 children in 2001 to 105 children in 200913. A national level study in Bangladesh in 2013 using a community level approach found prevalence of autism to be 0.15% amongst a population of 7200 in seven upazilas (Debhhata, Wazirpur, Pirgong, Godagari, Pekua, Madhupur and Kulaura and a city corporation ward of Dhaka city)14. In another study by the ministry of Social Welfare, Bangladesh 2016, the proportion of autism was found to be 19% of total neurological disabilities recorded15,16.\n\nIt is essential to identify the cases of ASD as early as possible because educational planning and initiation of interventions results in better outcomes for these children17–19. There are different methodologies applied in different studies to identify prevalence in Bangladesh. However, age specific prevalence of autism is not yet determined in Bangladesh. This study has explored the age specific (18–36 months) ASD prevalence among children from a rural community of Bangladesh.\n\n\nMethods\n\nA cross sectional study was conducted during the period of April 2016 to June 2016.\n\nRaiganj upazila (sub-district) of Sirajganj district is located in the northern part of Bangladesh. The upazila consists of nine unions (small unit of upazila) with a population of more than 300,000. The study implementing organization Center for Injury Prevention and Research, Bangladesh (CIPRB) has its own ongoing surveillance system functioning in six unions of this upazila. Thus, the study has chosen rural community of all six unions for the study.\n\nA total number of 255,265 populations reside in 55,492 households of the six unions of Raiganj upazila in Sirajganj district. All households of the selected unions were included for this study.\n\nThe study selected all children aged from 18 months to 36 months. Their information was recruited from the household database of the six unions’ surveillance system. A total number of 5600 children were identified from the surveillance data base. All households of those children were selected for collection of data at the household level (Figure 1).\n\nExpected outcome: The study looked for autism spectrum disorder as outcome and exposure was children of age 18–36 months either boys and girls in rural community.\n\nFifteen field level survey data collectors were recruited. Three supervisors were assigned to supervise and monitor the survey data collectors throughout the process of data collection and to confirm positive screened cases at the household level. All field level staff received one-day comprehensive training on data collection, which was conducted by a team of paediatric neurologists, public health specialists and child development therapists. The data collectors and supervisors were trained on using pictorial flash cards, which they were instructed to use during the time of interview to collect desired information (examples of flash cards are available in Supplementary File 1).\n\nThe data collectors performed face to face interviews with the mother at the household level using a structured questionnaire (Supplementary File 2). M-CHAT (Modified Checklist for Autism in Toddlers) was used as the primary screening tool for ASD. The survey data collectors used pictorial flash cards for each of the 23 questions of M-CHAT to let the mother understand if their children have similar type of manifestations as shown in the pictorial flash cards.\n\nA total number of 98 primarily screened positive cases were listed by the survey data collectors using M-CHAT. Out of 5600 children, the data collectors were able to collect data from 5286 children aged between 18 months to 36 months. Remaining families had either migrated from the unions or were not found during the data collection period. No respondent refused to provide information. The study has included participants those who were identified as M-CHAT positive during screening at the households to invite in the health camp.\n\nThree supervisors visited the 98 households after initial identification of the positive screened cases by the survey data collectors. The same M-CHAT and flash cards were used by the supervisors to confirm the primary diagnosis. Out of 98 primary positive screened cases, 68 cases were identified as M-CHAT positive cases. 30 cases were primarily screened out as MCHAT positive by the data collectors, did not match with the M-CHAT criterion during the 2nd visit by the supervisors. Before the exclusion of the 30 cases, investigators checked the MCHAT collected by the data collectors and the supervisors through a discussion meeting. Finally, 68 positive screened families were invited with their children to attend the medical camp for final diagnosis. Health camp was organized at CIPRB’s Raiganj research field office located in Dhangora union on 31st May 2016. Additionally, the investigators were involved in the process of quality assurance throughout the whole procedure of participation in trainings, data collection. They also randomly selected 5% of positive cases to check the consistency of data collection.\n\nBias and confounders: The study identified bias in identification of M-chat positive by the field level data collectors using pictorial flash cards. Initially, 98 children were identified as M CHAT positive. To prevent potential bias, all 98 children with positive M CHAT were visited by skilled supervisors. 68 cases finally identified M-CHAT positive, remains which were found M Chat negative, the supervisors sit with the research investigators to come up on final discussion to exclude from the study. The study did not address for any confounders.\n\nI. M-CHAT: It is a validated tool for assessing the risk of ASD in screening toddlers aged between 16 to 36 months. The M-CHAT can be administered and scored as part of a well-child check-up, and also can be used by specialists or other professionals to assess risk for ASD. Users need to be aware that even with the follow-up questions, a significant number of the children who fail the M-CHAT will not be diagnosed with an ASD. However, these children are at risk for other developmental disorders or delays, and therefore, evaluation are warranted for any child who fails the screening. Children who fail more than 3 items total or 2 critical items has been identified as initial screening positive for further diagnostic evaluation by professional experts to evaluate ASD in very young children20,21.\n\nII. DSM- IV TR: Diagnostic and Statistical Manual of Mental Disorders (DSM-IV TR), published by the American Psychological Association, is the standard for the classification of mental disorders. As ASD became known throughout the United States, and common symptoms and behaviors were agreed upon by many researchers, it gained increasingly specific diagnostic criteria in the DSM. Here, autism is traced throughout the four main domains of the DSM22.\n\nIII. ADOS: The Autism Diagnostic Observation Schedule (ADOS) is an instrument for diagnosing and assessing autism. It became commercially available in 2001 through the Western Psychological Services (WPS). The protocol consists of a series of structured and semi-structured tasks that involve social interaction between the examiner and the subject. The examiner observes and identifies segments of the subject's behavior and assigns these to predetermined observational categories. Categorized observations are subsequently combined to produce quantitative scores for analysis23.\n\nIV. Flash card: Twenty-three pictorial flash cards were developed based on 23 questions for M-CHAT to use in this study. All flash cards were drawn in a pictorial format. Flash cards were used during the survey at the household and shown each of the sign/symptoms in a pictorial form for better understanding for the mothers.\n\nA medical camp was organized at the upazila level to confirm the diagnosis and management of affected children. Out of M-CHAT positive 68 children, 66 children with their parents came to health camp for diagnosis. A team of two paediatric neurologists, three medical doctors experienced in working with children with autism, two child clinical psychologists, one development therapist from Institute of Paediatric Neurodisorder and Autism (IPNA), Bangabandhu Sheikh Mujib Medical University (BSMMU) along with a public health specialist (epidemiologist) conducted the health camp. The diagnosis and treatment were performed on three groups of children. The first group were all diagnosed with the DSM-4 TR, suspected ASD positive cases were then sent to the child clinical psychologist group for confirmation of ASD using the ADOS test. Finally, the third group provided management for all children including those who were identified as ASD or other NDDs (Figure 2).\n\nThree medical doctors, including one senior doctor, were involved in providing treatment for the children according to the diagnosis made by the paediatric neurologists. Management includes prescribing medicine, counseling, provision of physical therapy and emergency referral to a nearby referral centre of the district or IPNA of BSMMU.\n\nSPSS version 20 for windows was used for descriptive analysis. The prevalence and the confidence intervals were calculated using the software EPI Info Version 6.04d.\n\nEthical permission for this study was obtained from the Institutional Review Committee, Centre for Injury Prevention and Research, Bangladesh [Memo: CIPRB/ERC/2016/009]. Participants of the study were children aged between 18–36 months, to obtain details information about the children, written consent was obtained from the each of the parents to participate and provide information.\n\n\nResults\n\nThe majority of the mothers were aged between 18 and 30 years (74.2%). Most of them were had a low level of education or had no formal education (56.1%). The majority of the children were diagnosed between the age of 31 and 36 months (41%). Boys had found higher prevalence of diseases then girls (65% vs 55%) [Table 1].\n\nAmong the 66 children of rural community of Raiganj uapzila, three children were found to with no disease (4.5%). Several children were identified to have cerebral palsy (45.5%), whereas autism was diagnosed in only 6.1% of cases (n=4). Developmental delay was found as the second highest disease amongst the children (21.2%) (Figure 3).\n\nFrom the total sample (n=5286), only 4 cases were found to have ASD with a prevalence of 0.75/1000 children. Among the four cases three were boys and one was a girl, and their age ranged from 20–30 months. The highest prevalence rate found was for cerebral palsy at 5.6/1000 children. Developmental delay (2.6/1000) was the second highest prevalence (Figure 4).\n\nOut of 66 children, three children were diagnosed with no disease and no treatment or advice was suggested for them. For the remaining 63 children, 10% of the children were directly referred to the specialized centre for further management. A combination of medication, counseling and referral were advised for around 32% of children (Figure 5).\n\n\nDiscussion\n\nThe study found that the prevalence of autism is 0.75/1000 in rural children aged between 18–36 months, with prevalence of cerebral palsy and developmental delays being much higher. Age specific early diagnosis of autism in the rural community was not explored earlier. A national study conducted in 2013 showed that the overall prevalence of ASD was 1.55/1000 amongst the study group of 7280 children aged between 0–9 years. The study also revealed that prevalence of ASD in the rural community studied was 0.68/100014. Although it was estimated that about 300,000 children were affected with autism in Bangladesh with one case in every 94 boys, and one in every 150 girls, was estimated to suffer from ASD (see Autistic Children’s Welfare Foundation site). These findings are consistent with the present study. In the study by ministry of Social welfare of Bangladesh prevalence came out as 3% of total population. But in the study all the age groups were included15.\n\nThis study examined early detection of autism in children considering that the major symptoms of autism can be identified at this age group (2–3 years), and proper management as well as improved quality of life can be achieved from early detection24. ASD is usually described as a childhood neuro-developmental disorder with onset usually before 3 years of age25.\n\nThis study found that 56.1% of the mothers of ASD suspected children had low level or no formal education background. Another study also found the highest prevalence of ASD amongst children whose mothers only attended primary schools26. Contrary to this one study found children of parents with a higher educational background had a higher prevalence of childhood autism27.\n\nThis study also revealed that 74.2% mothers of suspected cases were aged between 18 to 30 years, with mothers with an early age of pregnancy had the higher rate of autistic children. This is in contrast with the study where advancing maternal age had found to be associated with higher risk of autism28. We also found autism to be more prevalent in boys than girls (65% vs 55%). A study in the US also revealed similar findings, where one in 42 boys and one in 189 girls were found with ASD29.\n\nA number of good examples were found on the estimation of prevalence of autism from the perspective of developed countries. Some countries used DSM-III for diagnostic purposes whereas others used ADOS30,31. Bangladesh has trialed for the first-time a community based system in the detection of autism among rural and urban communities through three stages of data collection, processing, and confirmation of cases15. This study used a community based population level survey at the household using pictorial flip cards to screen the cases for the first time. The study also used a four-stage data collection and confirmed diagnosis using a community based health camp. The study also followed the pathway of diagnosis to management, which creates a boarder spectrum of benefits for the early detection of autism and other neuro developmental diseases, as well as referral to the higher centers like ’child development center’ run by the Government of Bangladesh in different Medical Colleges and IPNA, BSMMU. Moreover, during management, each of the parents were counseled by the doctors who could help to improve overall quality of care for those children with autism and other neuro developmental disorders.\n\nIt is alarming that other neuro developmental diseases like cerebral palsy and developmental delays are many folds higher than autism. It is important to do further research on other NDDs so that special interventions can be designed based on the findings.\n\nThe study has been done at the sub district level within a confined population which may not be representative of the country. For better understanding of the real magnitude of the problem, a larger study is required, in a bigger population considering early age detection of ASD and other NDDs. Move over, it is also important to do further community based studies using the new approach adopted for primary diagnosis.\n\nThe country is well ahead for the reduction of under-five and infant mortality rate32. The country also has a very structured community level health infrastructure and primary health care delivery system, where community clinics have delivered health care services to people’s doors. It is time to respond on early detection of the ASD and other NDDs using the primary health care model. Although the definite causes of ASD are genetic, environmental modification and improvement of the quality for health care will improve the overall situation. Like this, early detection of ASD and NDDs will help the parents and their family to take immediate care for their better healthy quality of life.\n\n\nData availability\n\nThe study involved multiple stakeholders including government, professional organizations and research institute. Data is stored at the CIPRB and in Institute of Paediatric Neurodisorder and Autism (IPNA), Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka. Due to sensitivity of the data (contains identifying information), permission is required from the ethical committee for sharing data with a third party. Data requests should be sent to Institute of Paediatric Neurodisorder and Autism (IPNA), Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka who will contact the ethical review committee to gain approval to share the data. The conditions for gaining data access are a formal request with a clear objective and formal permission from the ethical committee. Please contact the Prof. Shaheen Akhter, Institute of Paediatric Neurodisorder and Autism (IPNA), Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka through email : shaheenk33@gmail.com in order to request the data.", "appendix": "Author contributions\n\n\n\nAuthors SA, EH, FR and AB designed this study. Authors SA, JS, GKK and AB reviewed literatures, analyzed surveyed data and prepared the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe study was financially supported by the Non Communicable Disease Control (NCDC), Directorate General of Health Services (DGHS), Mohakhali, Dhaka, Bangladesh.\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 gratefully acknowledge the contribution of Non Communicable Disease Control (NCDC), Directorate General of Health Services (DGHS), Mohakhali, Dhaka, Bangladesh for this study.\n\n\nSupplementary material\n\nSupplementary File 1 – Zip file contain examples of the flash cards used in this study\n\nClick here to access the data.\n\nSupplementary File 2 – Study questionnaire (with English translation)\n\nClick here to access the data.\n\n\nReferences\n\nPosar A, Visconti P: Autism in 2016: the need for answers. J Pediatr (Rio J). 2017; 93(2): 111–119. PubMed Abstract \| Publisher Full Text\n\nChristensen DL, Baio J, Van Naarden Braun K, et al.: Prevalence and Characteristics of Autism Spectrum Disorder Among Children Aged 8 Years--Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2012. MMWR Surveill Summ. 2016; 65(3): 1–23. PubMed Abstract \| Publisher Full Text\n\nInglese MD: Caring for children with autism spectrum disorder. Part II: screening, diagnosis, and management. J Pediatr Nurs. 2009; 24(1): 49–59. PubMed Abstract \| Publisher Full Text\n\nNevison CD: A comparison of temporal trends in United States autism prevalence to trends in suspected environmental factors. Environ Health. 2014; 13: 73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElsabbagh M, Divan G, Koh YJ, et al.: Global prevalence of autism and other pervasive developmental disorders. Autism Res. 2012; 5(3): 160–79 . PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHossain MD, Ahmed HU, Jalal Uddin MM, et al.: Autism Spectrum disorders (ASD) in South Asia: a systematic review. BMC Psychiatry. 2017; 17(1): 281. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlacher J, Christensen L: Sowing the seeds of the autism field: Leo Kanner (1943). Intellect Dev Disabil. 2011; 49(3): 172–91. PubMed Abstract \| Publisher Full Text\n\nNeggers YH: Increasing prevalence, changes in diagnostic criteria, and nutritional risk factors for autism spectrum disorders. ISRN Nutr. 2014; 2014: 514026. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCroen LA, Najjar DV, Fireman B, et al.: Maternal and paternal age and risk of autism spectrum disorders. Arch Pediatr Adolesc Med. 2007; 161(4): 334–40. PubMed Abstract \| Publisher Full Text\n\nSun X, Allison C: A review of the prevalence of Autism Spectrum Disorder in Asia. Res Autism Spectr Disord. 2010; 4(2): 156–67. Publisher Full Text\n\nMullick MS, Goodman R: The prevalence of psychiatric disorders among 5–10 year olds in rural, urban and slum areas in Bangladesh: an exploratory study. Soc Psychiatry Psychiatr Epidemiol. 2005; 40(8): 663–71. PubMed Abstract \| Publisher Full Text\n\nRabbani MG, Alam MF, Ahmed HU, et al.: Prevalence of mental disorders, mental retardation, epilepsy and substance abuse in children. Bangladesh J Psychiatry. 2009; 23(1): 1–54. Reference Source\n\nWilliams JG, Higgins JP, Brayne CE: Systematic review of prevalence studies of autism spectrum disorders. Arch Dis Child. 2006; 91(1): 8–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNCDC, RCHCIB, BMRC, et al.: Survey of Autism and Neurodevelopmental Disorders in Bangladesh, 2013. Non-Communicable Diseases Control (NCDC) Programme, DGHS, MOHFW, Revitalization of Community Health Care Initiatives in Bangladesh (RCHCIB), Ministry of health and family welfare (MOHFW); Bangladesh Medical Research Council (BMRC), MOHFW; Department of Pediatric Neuroscience, Dhaka Shishu Hospital, Dhaka, Bangladesh. 2013. Reference Source\n\nDisabilities Screening Bulletin. Ministry of Social Welfare of Bangladesh. 27 July, 2016.\n\nHaque KR: Addressing Autism and Disability: Making Progress in Bangladesh. 2015.\n\nFilipek PA, Accardo PJ, Baranek GT, et al.: The screening and diagnosis of autistic spectrum disorders. J Autism Dev Disord. 1999; 29(6): 439–84. PubMed Abstract \| Publisher Full Text\n\nNational Institute of Mental Health (NIMH): NIH Funds New Program to Investigate Causes and Treatment of Autism. NIMH. 2007. Reference Source\n\nNational Academy of Sciences & National Research Council: Educating children with autism. 2001. Publisher Full Text\n\nRobins DL, Fein D, Barton M: M-CHAT. 1999. Reference Source\n\nRobins DL, Fein D, Barton M: Modified Checklist for Autism in Toddlers, Revised with Follow-Up (M-CHAT-R/F). 2009; 1–25. Reference Source\n\nAmerican Psychiatric Association: Cautionary statement. Diagnostic and statistical manual of mental disorders (4th ed., text rev.). 2000.\n\nLord C, Risi S, Lambrecht L, et al.: The autism diagnostic observation schedule-generic: a standard measure of social and communication deficits associated with the spectrum of autism. J Autism Dev Disord. Los Angeles: Western Psychological Services, 2000; 30(3): 205–23. PubMed Abstract \| Publisher Full Text\n\nScherzer AL, Chhagan M, Kauchali S, et al.: Global perspective on early diagnosis and intervention for children with developmental delays and disabilities. Dev Med Child Neurol. 2012; 54(12): 1079–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoucas T, Charman T, Pickles A, et al.: Autistic symptomatology and language ability in autism spectrum disorder and specific language impairment. J Child Psychol Psychiatry. 2008; 49(11): 1184–92. PubMed Abstract \| Publisher Full Text\n\nLuzhong R, Zongzhi D, Xu J: An analysis on positive of rate autism among preschool children in city. Chinese J Child Care. 2003; 2: 105–7. Reference Source\n\nWeiwu L, Li L, Chen R: Epidemiological investigation on autistic disorder in Fujian Province. Shanghai Arch Psychiatry. 2000; 12(1): 3–5. Reference Source\n\nSandin S, Hultman CM, Kolevzon A, et al.: Advancing maternal age is associated with increasing risk for autism: a review and meta-analysis. J Am Acad Child Adolesc Psychiatry. 2012; 51(5): 477–486.e1. PubMed Abstract \| Publisher Full Text\n\nChristensen DL, Baio J, Van Naarden Braun K, et al.: Prevalence and Characteristics of Autism Spectrum Disorder Among Children Aged 8 Years--Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2012. MMWR Surveill Sum. 2016; 65(3): 1–23. PubMed Abstract \| Publisher Full Text\n\nKawa S, Giordano J: A brief historicity of the Diagnostic and Statistical Manual of Mental Disorders: issues and implications for the future of psychiatric canon and practice. Philos Ethics Humanit Med. 2012; 7(1): 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun X, Allison C, Auyeung B, et al.: Comparison between a Mandarin Chinese version of the Childhood Autism Spectrum Test and the Clancy Autism Behaviour Scale in mainland China. Res Dev Disabil. 2014; 35(7): 1599–608. PubMed Abstract \| Publisher Full Text\n\nSayem AM, Nury AT, Hossain MD: Achieving the millennium development goal for under-five mortality in Bangladesh: current status and lessons for issues and challenges for further improvements. J Health Popul Nutr. 2011; 29(2): 92–102. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "32768", "date": "18 Apr 2018", "name": "Olalekan Uthman", "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 of the manuscript conducted a cross-sectional study to estimate the prevalence of Autism Spectrum Disorder among children in Bangladesh. The manuscript has potential to contribute to the body of knowledge in the field. The authors should be congratulated on bringing to light this important, but often neglected spectrum of medical conditions in resource-limited settings. The manuscript reads well and conclusion stated were not beyond the findings of this cross-sectional study.\n\nI have no major concerns in the conduct and reporting of this study:\nAbstract: ‘04’ should be changed to word at the start of that sentence. Table 1: column two, consider reporting the number and percentage in the bracket and change the column heading to ‘Number (%)’ Discussion: Consider adding study strengths and limitations Discussion: In addition, consider adding sub-heading to the discussion section, such as ‘Main findings, ‘Implications for policy and research’, ‘Study strengths and limitations’ Conclusion: consider also adding conclusion at the end of the discussion section\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": "35443", "date": "27 Jun 2018", "name": "Helen McConachie", "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 cross-sectional study makes an estimate of the prevalence of autism spectrum disorder in very young children (18-36 months) in Bangladesh. The study utilised the opportunity of an ongoing rural surveillance system to carry out door to door screening using the M-CHAT tool enhanced by the use of flash-cards. Screen-positive status was checked by supervisors, and around two -thirds confirmed. These children were invited for diagnostic assessment, including where indicated assessment using a gold-standard diagnostic tool, the Autism Diagnostic Observation Schedule. A prevalence estimate of 0.75/1000 was concluded.\nThe paper cites the most relevant literature, including a 2013 Survey of Autism and Neurodevelopmental Disorders in Bangladesh conducted for the Directorate General of Health Services, Ministry of Health and Family Welfare, Government of Bangladesh. That survey found a very similar prevalence estimate of ASD in rural children of 0.68/1000. It is therefore incorrect for the authors to state in the Abstract: \"Age specific autism (18-36 months) in children in found higher in rural community of Bangladesh\". The estimate is low in comparison globally.\nThe methodology adopted for the study appears strong in the main. However, a prevalence estimate with confidence intervals would require that a random sample of children screened negative were also invited for further assessment. Some of the interpretation of results is also inappropriate as statements are made, for example, about the numbers of children identified with cerebral palsy, without clearly stating that this estimate is within only the screened positive and assessed children.\nThe authors' conclusions are encouraging, that systems for early detection and management of neurodevelopmental disorders can now be used across Bangladesh, because of good primary health care and the establishment of Child Development Centres in the Medical College Hospitals.\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": [] }, { "id": "35553", "date": "28 Jun 2018", "name": "Soumyadeep Bhaumik", "expertise": [ "Reviewer Expertise research methodology", "evidence syntheses" ], "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 intends to estimate the prevalance of autism spectrum disorder in rural children of 18-36 months.It uses a cross-sectional study design for this purpose. Comments for consideration are : 1.The use of the word age-specific in the objective is not neessary in my opinion. 2. The manuscript would benefit if it follows the STROBE reporting guidelines for cross-sectional study. Such reporting will enable understanding of the methodology better for the purpose of critique and reproducibility. This is mandatory as per F1000 guidelines. 3. Please clarify if the criteria for selection of unions - was it all the rural unions in Raiganj Upazila or was it the functioning ongoing surviellence system. Are the three unions which were not included as rural classified as urban by the government ? It might be good to name the six unions included. 4. The manuscript mentions all households of selected union was included - how was the list of universe of households obtained ? 5. Not sure what the expected outcome sub-section within sampling is for - consider deletion. 6. Please provide reference to MCHAT anf flash card methods being used in this study in other similar study. I am not sure why DSM-IV TR or ADOS is explained in the manuscript . Consider discussing why the issues are different in different studies. 7.Explain how the study size was calculated - was it powered for gender disaggregation. Was it powered for measuring other neurological conditions described in the paper. The result thus provides many other things and there is need of clarity to understand this better. 9. Discussion needs a section on potential limiations of the study and ways that has been taken to address the bias. How do the results of the study compare to that in other SEARO countries. 10. The Abstract conclusion mentions \" Age specific autism (18-36 months) in children is found higher in rural community of Bangladesh. \" It is not clear higher in comparison to what ? The idea about needing more studies in future is generic. Please consider providing more specific information. 11. Some points mentioned in the discussion like the \"56.1% of the mothers of ASD suspected children had low level or no formal education background. \" are probably of no intepretive value considered this is not a RR or OR and the fact that only 4 cases were found. Same for other things in the discussion.\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?\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-424
https://f1000research.com/articles/7-142/v1	02 Feb 18	{ "type": "Research Note", "title": "Spatial band-pass filtering aids decoding musical genres from auditory cortex 7T fMRI", "authors": [ "Ayan Sengupta", "Stefan Pollmann", "Michael Hanke", "Stefan Pollmann", "Michael Hanke" ], "abstract": "Spatial filtering strategies, combined with multivariate decoding analysis of BOLD images, have been used to investigate the nature of the neural signal underlying the discriminability of brain activity patterns evoked by sensory stimulation -- primarily in the visual cortex. Reported evidence indicates that such signals are spatially broadband in nature, and are not primarily comprised of fine-grained activation patterns. However, it is unclear whether this is a general property of the BOLD signal, or whether it is specific to the details of employed analyses and stimuli. Here we performed an analysis of publicly available, high-resolution 7T fMRI on the response BOLD response to musical genres in primary auditory cortex that matches a previously conducted study on decoding visual orientation from V1. The results show that the pattern of decoding accuracies with respect to different types and levels of spatial filtering is comparable to that obtained from V1, despite considerable differences in the respective cortical circuitry.", "keywords": [ "musical-genre decoding", "7 Tesla fMRI", "primary auditory cortex", "spatial band-pass filtering" ], "content": "Introduction\n\nWe recently reported1 that spatial band-pass filtering of 7 Tesla BOLD fMRI data boosts accuracy of decoding visual orientations from human V1. We observed this result in comparison to data without any dedicated spatial filtering applied, and spatially low-pass filtered data – a typical preprocessing strategy for BOLD fMRI. This effect was present across a range of tested spatial acquisition resolutions, ranging from 0.8 mm to 2 mm isotropic voxel size (Figure 4 in 1). Analysis of individual spatial frequency bands indicated the presence of orientation-related signal in a wide range of spatial frequencies as indicated by above-chance decoding performance for nearly all tested bands. Maximum decoding performance was observed for a band equivalent to a difference-of-Gaussians (DoG) filter of 5–8 mm full width at half maximum (FWHM), indicating that low spatial frequency fMRI components also contribute to noise with respect to orientation discrimination.\n\nThis finding raises the question whether this reflects a specific property of early visual cortex and the particular stimuli used in 1, or whether it represents a more general aspect of BOLD fMRI data with implications for data preprocessing of decoding analyses. Here, we investigate this question by applying the identical analysis strategy from 1 to a different public 7 Tesla BOLD fMRI dataset2, with the aim of decoding the musical genres of short audio clips from the early auditory cortex.\n\n\nMethods\n\nAs this study aims to replicate previously reported findings, by employing a previously published analysis strategy on an existing dataset, the full methodological details are not repeated here. Instead the reader is kindly referred to 2, 3 for comprehensive descriptions of the data, and to 1 for details on the analysis strategy and previous findings. Only key information and differences are reported below.\n\n\nStimulus and fMRI data\n\nData were taken from a published dataset2 which were repeatedly analyzed previously4,5, and publicly available from the studyforrest.org project of 20 participants passively listening to five natural, stereo, high-quality music stimuli (6 s duration; 44.1 kHz sampling rate) for each of five different musical genres: 1) Ambient, 2) Roots Country 3) Heavy Metal, 4) 50s Rock’n’Roll, and 5) Symphonic, while fMRI data were recorded in a 7 Tesla Siemens scanner (1.4 mm isotropic voxel size, TR=2 s, matrix size 160×160, 36 slices, 10% interslice gap). fMRI data were scanner-side corrected for spatial distortions6. Stimulation timing and frequency were roughly comparable to 1: 25 vs. 30 trials per run, 10 s vs. 8 s minimum inter-trial stimulus onset asynchrony in a low event-related design, 8 vs. 10 acquisition runs. Subject 20 was excluded from the analysis due to incomplete data.\n\n\nRegion of interest (ROI) localization\n\nAnalogous to 1, ROIs were localized separately for each individual brain. ROIs were left and right transversetemporal gyri, as defined by the structural Desikan-Killiany atlas7 from the previously published Freesurfer-based cortex parcellations for all studyforrest.org participants3. This ROI approximates the location of primary auditory cortex, including Broadmann areas 41 and 42 (Figure 1A). The average number of voxels in the ROI across participants was 1412 (std=357).\n\n(A) Localization of early auditory cortex (transversetemporal gyrus) as shown in coronal slice of a participant (sub-16). (B) Confusion matrix showing the mean performance of the LinearCSVM classifier across all participants in decoding musical genres from early auditory cortex in spatially unfiltered data. (C) Classification accuracy of decoding musical genres across different types and levels of spatial gaussian filtering. The theoretical chance performance of 20% is shown by the dashed line. This figure has been generated from original analysis of the dataset made publicly available2 under the terms of the Public Domain Dedication and License.\n\n\nfMRI data analysis\n\nMotion-corrected and distortion-corrected BOLD images from the publicly available dataset2 were analyzed. Images for each participant, available from the dataset as the filename pattern of sub/BOLD/task002_run/bold_dico_bold7Tp1_to_subjbold7Tp1.nii.gz were already aligned across acquisition runs. Analogous to 1, BOLD images were masked to the defined bilateral ROI, and voxelwise BOLD response were univariately modelled for each run using the GLM implementation in NiPy [v0.3;8] while accounting for serial correlation with an autoregressive term (AR1). The GLM design matrix included hemodynamic response regressors, one for each genre and its corresponding temporal derivatives, six nuisance regressors for motion (translation and rotation), and polynomial regressors (up to 2nd-order) modeling temporal signal drift as regressors of no-interest. The β weights thus computed for each run were Z-scored per voxel. Multivariate decoding was performed on these Z-scored β weights using linear support vector machines [SVM; PyMVPA’s LinearCSVMC implementation of the LIBSVM classification algorithm;9,10] in a within-subject leave-one-run-out cross-validation of 5-way multi-class classification of musical genres. The hyper-parameter C of the SVM classifier was scaled to the norm of the data. Decoding was performed using the entire bilateral ROI.\n\nIn-line with 1, 11, complete BOLD images were spatially filtered prior masking and GLM-modeling, as prior results suggest negligible impact of alternative filtering strategies (see Figure S4 in 1). The magnitude of spatial filtering used is expressed in terms of the size of the Gaussian filter kernel(s) described by their FWHM in mm. The image_smooth() function in the nilearn package12 was used to implement all spatial smoothing procedures. The implementations of Gaussian low-pass (LP), and high-pass (HP) filters, as well as the DoG filters for bandpass (BP) and bandstop (BS) filtering are identical to those of 1 (1 mm FWHM filter size difference).\n\n\nResults and conclusions\n\nFigure 1 shows the mean accuracy across 19 participants for classifying the genre of music clips from BOLD response patterns of bilateral early auditory cortex. Compared to visual orientation decoding from V11, the mean accuracy of decoding musical genres without dedicated spatial filtering exhibits a substantially higher baseline (for 1.4 mm unfiltered data, mean orientation decoding accuracy was around 35%, whereas mean decoding of musical genres was at around 65%). However, the general pattern of accuracies across all filter sizes and filter types strongly resembles the results of orientation decoding from V1. LP filtering led to a steady decline of performance with increasing filter size, but does not reach chance level even with a 20 mm smoothing kernel. In contrast to LP filtering, HP filtered data yielded superior decoding results for filter sizes of 4 mm and larger. Congruent with 1, BP filtering led to maximum decoding accuracy in the ≈5-8 mm FWHM band. The accuracy achieved on BP filtered data at 6mm FWHM was significantly higher than that without any dedicated spatial filtering (McNemar test with continuity correction13: χ2=33.22, p<10–6). BS filtering led to an approximately constant performance regardless of the base filter size, on the same level as with no dedicated spatial filtering.\n\nIn line with Gardumi et al.14, these results suggest that BOLD response patterns informative for decoding musical genre from early auditory cortex are spatially distributed and are represented at different spatial scales. However, despite their broadband nature, relevant information seems to be concentrated in the spatial frequency band corresponding to a ≈5–8 mm DoG filter. Most notably, the present findings show a striking similarity to the visual orientation decoding accuracy patterns in V11.\n\nThe origin and spatial scale of signals beneficial for decoding BOLD response patterns are an intensely debated topic in the literature, and various studies have looked at this question in the context of anatomical or topographical structure of visual cortex11,15–17.\n\nThere are substantial differences between the auditory and visual cortex in terms of anatomy, synaptic physiology, and the circuity of cortical layers and their connections with other cortical areas and subcortical nuclei18. The present results indicate that these differences have little impact on the spatial characteristics of those BOLD signal components that are relevant for decoding visual orientation or genre of music. In summary, these findings call for further investigations of neural and physiological signals underlying decoding models that are common across sensory domains, and individual cortical areas. The increasing availability of diverse open brain imaging data can help to aid the evaluation of generality and validity of explanatory models.\n\n\nData and software availability\n\nOpenFMRI.org: High-resolution 7-Tesla fMRI data on the perception of musical genres. Accession number: ds000113b\n\nArticle sources for 7-Tesla fMRI data on the perception of musical genres are available: https://doi.org/10.5281/zenodo.1876719\n\n“Forrest Gump” data release source code is available: https://doi.org/10.5281/zenodo.1877020\n\nThe codes used in this study for analysis are made openly available: https://doi.org/10.5281/zenodo.115883621", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAS and SP were supported by a grant from the German Research Concil (DFG) awarded to S. Pollmann (PO 548/15-1), MH was supported by funds from the German federal state of Saxony-Anhalt and the European Regional Development Fund (ERDF), Project: Center for Behavioral Brain Sciences. This research was, in part, also supported by the German Federal Ministry of Education and Research (BMBF) as part of a US-German collaboration in computational neuroscience (CRCNS; awarded to J.V. Haxby, P. Ramadge, and M. Hanke), co-funded by the BMBF and the US National Science Foundation (BMBF 01GQ1112; NSF 1129855).\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\nSengupta A, Yakupov R, Speck O, et al.: The effect of acquisition resolution on orientation decoding from V1 BOLD fMRI at 7T. Neuroimage. 2017; 148: 64–76. PubMed Abstract \| Publisher Full Text\n\nHanke M, Dinga R, Häusler C, et al.: High-resolution 7-Tesla fMRI data on the perception of musical genres – an extension to the studyforrest dataset [version 1; referees: 2 approved with reservations]. F1000Res. 2015; 4: 174. Publisher Full Text\n\nHanke M, Baumgartner FJ, Ibe P, et al.: A high-resolution 7-Tesla fMRI dataset from complex natural stimulation with an audio movie. Sci Data. 2014; 1: 140003. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGüçlü U, Thielen J, Hanke M, et al.: Brains on beats. In Advances in Neural Information Processing Systems. 2016; 2101–2109. Reference Source\n\nCasey MA: Music of the 7Ts: Predicting and Decoding Multivoxel fMRI Responses with Acoustic, Schematic, and Categorical Music Features. Front Psychol. 2017; 8: 1179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIn MH, Speck O: Highly accelerated PSF-mapping for EPI distortion correction with improved fidelity. MAGMA. 2012; 25(3): 183–192. PubMed Abstract \| Publisher Full Text\n\nDesikan RS, Ségonne F, Fischl B, et al.: An automated labeling system for subdividing the human cerebral cortex on MRI scans into gyral based regions of interest. Neuroimage. 2006; 31(3): 968–980. PubMed Abstract \| Publisher Full Text\n\nMillman KJ, Brett M: Analysis of functional magnetic resonance imaging in Python. Comput Sci Eng. 2007; 9(3): 52–55. Publisher Full Text\n\nChang CC, Lin CJ: LIBSVM: A library for support vector machines. ACM Trans Intell Syst Technol. 2011; 2(3): 27. Publisher Full Text\n\nHanke M, Halchenko YO, Sederberg PB, et al.: PyMVPA: A Unifying Approach to the Analysis of Neuroscientific Data. Front Neuroinform. 2009; 3: 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwisher JD, Gatenby JC, Gore JC, et al.: Multiscale pattern analysis of orientation-selective activity in the primary visual cortex. J Neurosci. 2010; 30(1): 325–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPedregosa F, Varoquaux G, Gramfort A, et al.: Scikit-learn: Machine learning in Python. J Mach Learn Res. 2011; 12: 2825–2830. Reference Source\n\nEdwards AL: Note on the correction for continuity in testing the significance of the difference between correlated proportions. Psychometrika. 1948; 13(3): 185–187. PubMed Abstract \| Publisher Full Text\n\nGardumi A, Ivanov D, Hausfeld L, et al.: The effect of spatial resolution on decoding accuracy in fmri multivariate pattern analysis. Neuroimage. 2016; 132: 32–42. PubMed Abstract \| Publisher Full Text\n\nFreeman J, Brouwer GJ, Heeger DJ, et al.: Orientation decoding depends on maps, not columns. J Neurosci. 2011; 31(13): 4792–804. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlink A, Krugliak A, Walther A, et al.: fMRI orientation decoding in V1 does not require global maps or globally coherent orientation stimuli. Front Psychol. 2013; 4: 493. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreeman J, Heeger DJ, Merriam EP: Coarse-scale biases for spirals and orientation in human visual cortex. J Neurosci. 2013; 33(50): 19695–703. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLinden JF, Schreiner CE: Columnar transformations in auditory cortex? A comparison to visual and somatosensory cortices. Cereb Cortex. 2003; 13(1): 83–89. PubMed Abstract \| Publisher Full Text\n\nHanke M: paper-f1000_pandora_data: Initial submission (Version submit_v1). Zenodo. 2015. Data Source\n\nHanke M, v-iacovella, Häusler C: gumpdata: Matching release for pandora data paper publication (Version pandora_release1). Zenodo. 2015. Data Source\n\nSengupta A: psychoinformatics-de/studyforrest-paper-auditorydecoding: v1.0 (Version v1.0). Zenodo. 2018. Data Source" }	[ { "id": "30541", "date": "08 Feb 2018", "name": "Cyril R. Pernet", "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 note, replicating previous results from vision to audition - and therefore showing that band-pass is an effective strategy, not specific to visual columns.\n\nMethod:\nYou set a GLM to get beta estimates per genre for each run, and use this (z-scored) for decoding. You mention that you included temporal derivatives, were these ones used in any form? (note you can also correct the hrf amplitude estimates using the temporal derivative https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896880/)\n\nYou used a within-subject leave-one-run-out cross-validation; as you know I'm sure, Gael showed that LOO tends to be biased (https://arxiv.org/abs/1606.05201). If I understand correctly, that means that the classification is done with 7 betas per class, predict 1 (and rotate). Obviously, that is an issue as K-folds will lead to an even smaller number of beta to use. I don't know if that's addressable here, but worth mentioning the issue (and even better do an alternative sampling scheme if you think it's feasible).\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? 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": [ { "c_id": "3555", "date": "04 Apr 2018", "name": "Ayan Sengupta", "role": "Author Response", "response": "This is a good note, replicating previous results from vision to audition - and therefore showing that band-pass is an effective strategy, not specific to visual columns. Method: You set a GLM to get beta estimates per genre for each run, and use this (z-scored) for decoding. You mention that you included temporal derivatives, were these ones used in any form? (note you can also correct the hrf amplitude estimates using the temporal derivative https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896880/) The GLM was performed in NiPy [v0.3;8] while accounting for serial correlation with an autoregressive term (AR1) and hrf modelling was set to be ‘canonical with derivative’. The temporal derivatives were included in the model for a better estimation of the beta parameters. This is now explicitly mentioned in the manuscript with corresponding citation. You used a within-subject leave-one-run-out cross-validation; as you know I'm sure, Gael showed that LOO tends to be biased (https://arxiv.org/abs/1606.05201). If I understand correctly, that means that the classification is done with 7 betas per class, predict 1 (and rotate). Obviously, that is an issue as K-folds will lead to an even smaller number of beta to use. I don't know if that's addressable here, but worth mentioning the issue (and even better do an alternative sampling scheme if you think it's feasible). We thank the reviewer for raising this issue with the cross-validation procedure. We agree that the leave-one-out (LOO) cross validation that was performed in this analysis may be biased and repeated random sub-sampling of training set could address the issue. However it has to be noted that the LOO cross validation method was almost exclusively used in all auditory and visual decoding in prior publications. As rightly mentioned by the reviewer, this study is about ‘replicating previous results from vision to audition’ and hence we keep on performing leave-one-out cross validation for making a direct comparison. The caveats of LOO cross validation are now mentioned in the results and conclusion section." } ] }, { "id": "30542", "date": "09 Mar 2018", "name": "David G. Norris", "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 explores the effect of spatial filtering on the decoding accuracy of BOLD fMRI data. The authors conclude that a spatial bandpass filter corresponding to a difference of Gaussians of 6mm improves the decoding accuracy.\nAbstract: 'Reported evidence' is clumsy phrasing how about something like 'previous research'. Use of 'matches' is also ambivalent: do you mean the analysis was similar or the results? Please state the main result and not just that it is similar to that obtained for V1.\nIntroduction: The term spatial frequency is used frequently, whereas the difference of Gaussians is described in terms of a FWHM. The units of spatial frequency are 1/mm, so if you are describing band pass filters, cut-offs etc in terms of spatial frequency then please convert to the correct units.\nStimulus and fMRI data: Please give slice orientation of fMRI acquisition. I believe it was axial, but Figure 1 could mislead people into thinking it was coronal.\nFigure 1 caption: gaussian->Gaussian.\nfMRI data analysis: prior masking -> prior to masking\nResults and Conclusions: These are given with little discussion. Why do you think the decoding accuracy is so much higher for music than for V1. Some discussion of existing literature would also be welcome, also of papers which do not reach the same conclusion as the authors (ref 1).\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? 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": "3554", "date": "04 Apr 2018", "name": "Ayan Sengupta", "role": "Author Response", "response": "This article explores the effect of spatial filtering on the decoding accuracy of BOLD fMRI data. The authors conclude that a spatial bandpass filter corresponding to a difference of Gaussians of 6mm improves the decoding accuracy. Abstract: 'Reported evidence' is clumsy phrasing how about something like 'previous research'. Use of 'matches' is also ambivalent: do you mean the analysis was similar or the results? Please state the main result and not just that it is similar to that obtained for V1. The required phrases have been changed accordingly. Introduction: The term spatial frequency is used frequently, whereas the difference of Gaussians is described in terms of a FWHM. The units of spatial frequency are 1/mm, so if you are describing band pass filters, cut-offs etc in terms of spatial frequency then please convert to the correct units. The unit of spatial frequency (FWHM) is kept unaltered in order to maintain parity with other previous publications. But a conversion of units to (cycles/mm) and the implementation of DoG filter is explained in Supplementary Figure 5 of Sengupta et al. 2017. It is now explicitly referred to in the manuscript. Stimulus and fMRI data: Please give slice orientation of fMRI acquisition. I believe it was axial, but Figure 1 could mislead people into thinking it was coronal. The words ‘axial slices’ are now mentioned in the description of the acquisition protocol. Figure 1 caption: gaussian->Gaussian. The change is incorporated into the manuscript. fMRI data analysis: prior masking -> prior to masking This change is done. Results and Conclusions: These are given with little discussion. Why do you think the decoding accuracy is so much higher for music than for V1. Some discussion of existing literature would also be welcome, also of papers which do not reach the same conclusion as the authors (ref 1). We thank the reviewer for pointing this point in the manuscript. These points are now discussed in the results and conclusion section." } ] } ]	1	https://f1000research.com/articles/7-142
https://f1000research.com/articles/7-415/v1	29 Mar 18	{ "type": "Research Article", "title": "Potential of bacteriocins produced by probiotic bacteria isolated from tiger shrimp and prawns as antibacterial to Vibrio, Pseudomonas, and Aeromonas species on fish", "authors": [ "Feli Feliatra", "Zainal A. Muchlisin", "Hiwan Yuda Teruna", "Widya Rahmi Utamy", "Nursyirwani Nursyirwani", "Andi Dahliaty", "Feli Feliatra", "Hiwan Yuda Teruna", "Widya Rahmi Utamy", "Nursyirwani Nursyirwani", "Andi Dahliaty" ], "abstract": "Backgrounds: Bacteriocin has been used widely in industry as a biopreservative agent. The objective of the present study was to investigate the potency of Bacteriocin isolated from tiger prawn Penaeus monodon and freshwater shrimp Macrobrachium rosenbergii as an anti-bacterial on fish. Methods: A total of ten candidates of probiotic bacteria consisted of five isolates from tiger shrimps (H1, H2, H3, H4, H5) and five isolates from freshwater prawns (W1, W2, W3, W4, W5) were evaluated. Bacteriocin wasBacteriocin was produced by centrifugation at a speed of 150 rpm and at 37 °C for 24 hours. The bacteriocin extract was purified by adding sulphate ammonium salt {(NH4) 2SO4} at 80% of the saturation level. Bacteriocin activity was determined using a diffusion method against pathogenic bacteria Vibrio alginolyticus, Aeromonas hydrophillaAeromonas hydrophilla and Pseudomonas stutzeri. Bacteriocins were analyzed usinganalyzedusing High Performance Liquid Chromatography (HPLC) and Fourier Transform Infra-Red (FTIR). The data were subjected to analysis of variance (ANOVA) and followed with Duncans multiple range test. Results: Bacteriocins produced by bacteria isolate H4 from tiger prawn indicated the highest bacteriocin activity againstbacteriocin activity against Pseudomonas stutzeri atstutzeri at the diameter of inhibition zone of 887.10 ± 409.24 mm2/mL. While isolate W2 from freshwater shrimp indicated inhibition zone of 1466.96 ± 127.62 mm2/mL. Both bacteriocins were purified by chromatography column using Sephadex LH-20. Conclusions: This finding showed that bacterial isolates H4 and W2 have the potential to produce bacteriocins which inhibit the pathogenic bacteria. FTIR analysis showed an amide group at wave number 1652cm-1 contained in the bacteriocins of isolates H4 and W2.", "keywords": [ "potency", "bacteriocin", "probiotic", "tiger shrimp", "prawn", "antibacterial", "pathogen" ], "content": "Introduction\n\nProbiotics are beneficial microbes for living organisms and, in certain quantities, have a positive impact on health1,2. Probiotic bacteria play a vital role in suppressing the growth of pathogenic microbial populations. Probiotic bacteria including lactic acid bacteria have an ability to produce several antimicrobial compounds such as lactic acid, diacetyl, hydrogen peroxide, carbon dioxide and bacteriocins3,4.\n\nBacteriocins are antimicrobial peptides which act as antibacterial compounds against bacterial pathogens5. Bacteriocins can inhibit Gram positive and Gram negative bacteria such as Salmonella species (sp.), Escherichia coli, Vibrio sp., Shigella sp., Aeromonas sp. and Pseudomonas sp6. Bacteriocins have an important role against microbes as a bactericidal as they are resistant to heat and maintain activity in an acidic environment, and low temperature during storage does not affect bacteriocin activity5. Bacteriocins can be damaged by degradation from proteolytic enzymes7.\n\nLately the use of bacteriocins has become of interest due to their potential use as a preservative in the food industry, especially in fermented foods. Nisin, for example, is one of the bacteriocins produced by lactic acid bacteria of Lactococcuslactis that has been known and recognized as safe to use, and can degrade pathogenic and spoilage microbes. Therefore, it could improve the quality and shelf life of food products8. This study examines extracts of bacteriocins produced by probiotic bacteria isolated from tiger shrimp and prawn which have an ability to inhibit pathogenic bacteria. The findings in this study will help researchers to inform fish farmers in preparing food for fish and shrimp populations in order to prevent pathogens, improve food efficiency and to increases the animals’ immune system.\n\n\nMethods\n\nA total of ten isolates of probiotic bacteria were collected, consisting of five isolates from tiger shrimp and five isolates from prawn9, which were from the collection of the Marine Microbiology Laboratory of the Faculty of Fisheries, University of Riau, Indonesia. ID numbers for each isolates are (GenBank): H1: KY995544, H2: KY995545, H3: KY995546, H4: KY995547, H5: KY995548, W1: KY995549, W2: KY995550, W3: KY995551, W4: KY995552 and W5: KY995553. This study was conducted from April-September 2016. Pathogenic gram-negative bacteria, Vibrio alginolyticus was ontained from Brackish water Research Institute in Jepara, Indonesia, while Aeromonas hydrophila and Pseudomonas stutzeri were purchased from Quarantine Centre in Pekanbaru, Indonesia.\n\nThe Nutrient Agar (NA; Merck, Kenilworth, NJ, USA, Cat.No. 1.05450.0500), Nutrient Broth (NB; Thermo Scientific Oxoid, Waltham, MA, USA, Cat.No.CM0001), Thiosulfate Citrate Bile Sucrose (TCBS; Merck, Cat.No.1.10263.0500), Trypticase Soy Agar, (TSA;Merck, Cat.No. 1.05458.0500), Trypticase Soy Broth (TSB, Merck, Cat.No. 1.05459.0500) were used in this study.\n\nGlycerol, glasswool, paper disc 6mm (Macharey-Nagel, Düren, Germany, MN827ATD), gel filtration chromatography using Sephadex LH-20 (Sigma-Aldrich, St-Louis, MO, USA - Offer sigma-GE17-0090-01), methanol and other chemicals were used in accordance with the laboratory procedures.\n\nProbiotic bacteria can grow in acidic conditions so each process was performed in physiological acidic pH solutions. In this study, probiotic bacteria were grown at pH 4. The probiotic bacteria was cultured in the following manner: a tube filled with the 500μL physiological solution of pH 4 was inoculated with probiotic bacteria aseptically at concentration of 108 cfu/mL. The mixture was homogenized and left to stand for 5 minutes. One loop full of the mixture was streaked on NA media. Media containing the bacteria were incubated at 37 °C for ± 24 hours.\n\nLiquid medium, nutrient broth of 300 mL volume was used for the production of bacteriocins10. The Medium was sterilized in an autoclave at a pressure of 15 psi, at 121°C for 15 minutes. After one day at room temperature, the medium was inoculated with 5% of probiotic bacteria that had previously been incubated overnight at the optical density (OD) at 600nm ~ 0.1 (v/v) which was equivalent to 107 CFU/mL (OD measured with Thermo Scientific GENESYS 10S Uv-Visible)11. The inoculated media was then fermented in a shaking incubator at a speed of 150 rpm for ± 24 hours at 37 °C. After incubation, the fermented medium was cooled in a refrigerator at 5–10°C for ± one hour. The crude bacteriocin extract from the medium by centrifugation (Hitachi, Tokyo, Japan – CS150FNX) at 10,000 rpm for 10 min at 4°C. The supernatant was then separated by filtration through glasswool. The cell-free supernatant (extracted bacteriocins) produced, was tested for activity against pathogenic bacteria by using a disc diffusion method. Some of the supernatants was precipitated by the addition of 80% salt ammonium sulfate [(NH4) 2SO4], and then was tested for activity in the same manner, and finally was analyzed by using High-performance liquid chromatography (HPLC).\n\nBacteriocin was precipitated from the crude extract by the addition of 80% salt ammonium sulfate [(NH4) 2SO4] in a cold condition (temperatures of 5 °C to 10 °C) while stirring gently to achieve 80% saturation and was then left overnight. The precipitate was then separated from the filtrate by centrifugation (Hitachi – CS150FNX) in a cold state at 13,000 rpm for 10 minutes. After centrifugation, the precipitate was added with 0.05 M phosphate buffer solution at pH 7.0, and it was ready to be used for the bacteriocins activity test. The rest was put in microtubes and was stored in a freezer (-20 °C).\n\nBacteriocin activity was tested by the diffusion agar method by using a paper disc of 6 mm pore size against pathogenic bacteria V. alginolyticus, A. halophyla, and P. stutzeri. One mL of each of pathogen inoculum (OD 600nm ~ 0.1, which is equivalent to 107 CFU/mL11 was inoculated into a test tube containing 15 mL of NA medium and was then vortexed. The inoculated medium was poured into a Petri dish and was allowed to solidify. A total of 50 mL of bacteriocins (bacteriocin extract both before and after precipitation) was dropped on paper discs and was allowed to dry. Amoxsan® 30μg from local pharmacy in Pekanbaru Indonesia was used as positive control and sterile liquid medium as the negative control. The dried paper discs were placed on pathogenic-inoculated NA media. After incubation for ± 24 hours, the bacteriocin activities were indicated by clear zone formed around the discs. The diameter of the clear zones was measured by using calipers. Inhibitory activity against pathogenic bacteria of extracellular fluid was calculated as AU (Activity Unit). One AU/mL was the area of inhibition zone per unit volume of bacteriocinsamples tested (mm2/ml). Bacteriocin activity can be calculated using the following equation5:\n\nBacteriocin Activity (mm2/mL) = Lz–LcV\n\nWhere:\n\nLz = diameter of clear zone area (mm2/ml)\n\nLc = Disc diameter (mm2/ml)\n\nV = Sample Volume (mL)\n\nGel filtration or gel permeation is a protein separation technique based on molecular size12. This technique used a column measuring 2.5 × 50 cm (Sephadex LH-20) as steady phase and methanol as mobile phase. A total of 9 g Sephadex LH-20 was weighed and then was added to 50 mL of methanol. The mixture was stirred gently until dissolved. The dissolved Sephadex was poured into the column until the marked limit. The eluent was collected in a beaker containing a mixture of Sephadex, it was then poured again into the column until Sephadex expanded and solidified for 20 minutes. Afterward, the bacteriocin was inserted into the column through the column wall carefully and waited until the sample penetrated through pores of the gel Sephadex LH-20 pores. The eluate was then collected in a marked vial as the first fraction, and the next eluent was collected in vials containing 20 drops until final-clear eluent reached. Small molecules will enter the pores of the gel Sephadex and move slowly, while the large molecules will move faster because it cannot enter and retaining the gel. Thus, the large molecules will emerge as the initial component. Products of each of separated-bacteriocin fractions were finally analyzed by using FTIR spectroscopy.\n\nBacteriocins produced after precipitation in ammonium sulphate was then analyzed using HPLC. A spectrophotometer ultraviolet (UV) detector (Thermo Scientific Genesys 10S) was used for the analysis of bacteriocins at a wavelength of 210 nm and 250 nm. Wavelength selection was based on preliminary measurement using UV spectrophotometer in The Research Laboratory of Enzymes, Fermentation, and Biomolecular, following the research performed by Masuda et al.,13. The analysis used Shimadzu HPLC system of UFLC Shim Pack C18 series with column size of 4.6 mm × 250 mm (Shimadzu, Kyoto, Japan).\n\nThe data were subjected to one-way analysis of Variance (ANOVA)14 followed by Duncan multiple range testat significance levels of 95%. The analysis was performed using a SPSS ver.20 software.\n\n\nResults\n\nInhibitory activity of bacteriocins was expressed as the inhibition zone per unit volume of samples tested (mm2/mL). Table 1 shows the activity of bacteriocins extract of tiger shrimp against pathogenic bacteria V. alginolyticus, A. hydrophila and P. stutzeri. Statistical analysis indicated that the activity was not significantly different (P> 0.05), the highest activity was indicated by bacteria isolate H1 against V. alginolyticus. However, the activity was not significantly different (P> 0.05) from isolates H2 and H5, which was 674.65 mm2/mL, but it was significantly different (P> 0.05) from isolates H3 and H4 (Table 1). The highest activity of bacteriocin crude extract against A. hydrophila was found at 319.91 mm2/mL from isolate H4, which was not significantly different (P> 0.05) from isolates H2, H3, and H5. However, it was significantly different (P> 0.05) from isolate H1 (P <0.05). The highest inhibitory activity against P. stutzeri was indicated by isolate H5 which was significantly different (P <0.05) from four other bacteria.\n\nNote: H = Bacterial Code. Superscript of the same letters indicates no significant differences in the level of 5%. Values were average of triplicate samples ± standard deviation.\n\nAfter precipitation with ammonium sulphate [(NH4)2SO4], the activity of each of bacteriocins tends to change. The highest activity was performed by isolate H3 against V alginolyticus, that was 896.50 mm2/mL (Table 2). Meanwhile, the lowest activity was indicated by isolate H1 that was 321.47 mm2/mL. This condition was different from that which was before been precipitated with ammonium sulphate. The highest activity was indicated by isolate H1 against V alginolyticus (Table 1). This concluded that precipitation process resulted in the different effect on bacteriocin produced by each of tested bacteria.\n\nNote: H = Bacterial Code. Superscript of the same letters indicates no significant differences in the level of 5%. Values were average of triplicate samples ± standard deviation.\n\nThe activity of the bacteriocins-crude extract of bacterial isolates from prawns against pathogens V. alginolyticus, A. hydrophila and P. stutzeri was not significantly different (P>0.05), and the highest activity was indicated by isolate W4 against A. hydrophila (746.95 mm2/mL). In comparison to bacteriocin produced by bacteria isolated from black tiger shrimp, of which the highest was indicated by isolate H1 against A. hydrophila, that was 674.65 mm2/mL. Therefore, the activity of bacteriocin produced by bacteria from prawns was higher than that of black tiger shrimp.\n\nValues for W4 were not significantly different from isolates W2 and W5 (P>0.05), but it was significantly different from W1 and W3 (P <0.05). The highest activity of the bacteriocins-crude extract against V. alginolyticus was shown by isolate W4 (645.76 mm2/mL), which was significantly different (P<0.05) from other four isolates. Meanwhile, the highest activity of bacteriocins-crude extract against P. stutzeri was indicated by isolate W2 (412.73 mm2/mL), which was significantly different (P <0.05) from the other four isolates.\n\nBacteriocinactivities of bacterial isolates from prawns against all pathogens (V. alginolyticus, A. hydrophila, and P. stutzeri) were not significantly different (P>0.05). The highest activity was produced by isolate W2 against P. stutzeri, and the highest inhibitory activity was 1466.96 mm2/mL which was significantly (P> 0.05) different from other four bacteria isolates (Table 4). However, the activity of isolate W1 was not significantly different from isolate W5 (P> 0.05) against P. stutzeri (Table. 4). Overall, the activity of bacteriocin precipitated in ammonium sulphate {(NH4)2SO4} was higher than bacteriocin before precipitation in ammonium sulphate.\n\nA quantity of 270 mL of bacteriocin-crude extract was obtained after centrifugation, and then it was precipitated with ammonium sulphate salt [(NH4)2SO4]. In the deposition process at salt saturation level of 80%, a total of 139.32 g of salt ammonium sulphate [(NH4) 2SO4] was added to the crude extract of bacteriocins. The precipitated bacteriocin was added with buffer in order to reach a volume of 3,375 mL bacteriocins.\n\nHPLC analysis. High-Performance Liquid Chromatography (HPLC) is a chromatographic method that uses a reversed-phase system as its working system. This method was used to determine the purity level of a compound to be analyzed. Bacteriocins of tiger shrimp and prawns with high bacteriocin-activity values (H4 and W2), were then analyzed by HPLC as shown in Figure 1 and Figure 2. The figures showed that bacteriocin produced by each of probiotic bacteria was not a pure product indicated by the number of chromatogram peaks.\n\nBacteriocins as a group of proteins, were separated by column chromatography on Sephadex LH-20 using methanol. The resulted eluate was initially appeared light brown in color in the column, and was collected in a vial which was then marked as fraction 1. After that, as the sample was in the middle of the column, the next eluate was collected as fraction 2, and followed by collecting every 20 drop samples until the sample sappeared faded, and finally clear in vial 25. This indicates that the protein samples in the column Sephadex LH-20 have been completely eluted by methanol. The eluate of 25 fractions was allowed to evaporate at room temperature to remove the solvent. The solids obtained were observed to look like thin films in some of the fractions. Five fractions contained thin films of 25 fractions produced, those were fractions 6, 9, 14, 18 and 25, which were then analyzed using FTIR spectroscopy to observe the functional groups containing the fraction.\n\nFourier Transform Infra-Red (FTIR) was used to find out bonding vibration and functional groups contained by bacteriocins. FTIR analysis results from one of the bacteriocins produced by bacteria H4 are shown in Figure 3. The spectrum figure shows the comparison of fractions that produced films (fractions 6, 9, 14, 18 and 25) as the separation products of bacteriocins H4 using gel filtration column, and after being precipitated in ammonium sulphate [(NH4) 2SO4]. Each fraction showed absorption bands at similar wave numbers.\n\nThe infrared spectrum of bacteriocins for fraction 6 shows absorption at wave numbers (cm-1), 978, 1340, 1355, 1648, 2831, 2921, 3420 and 3595. Fraction 9 shows absorption at wave numbers (cm-1) of 977, 1089, 1339, 1363, 1404, 1457, 1647, 3239, and 3587. Fraction 14 showed absorption at wave numbers (cm-1) of 978, 1097, 1312, 1363, 1405, 1457, 1647, 2836, and 3502. Fraction 18 shows absorption at wave numbers (cm-1) of 988, 1334, 1358, 1400, 1460, 1649, 2835, 2954, 3415 and 3588. Finally, fraction 25 shows absorption at wave numbers (cm-1) of 971, 1082, 1312, 1339, 1355, 1416, 1457, 1647, 2834, 2988, 3336, 3443 and 3593. Meanwhile, bacteriocins precipitated by salts of ammonium sulphate [(NH4) 2SO4] shows an absorption band at wave number (cm-1) 979, 1339, 1355, 1418, 1456, 3336, 3445, and 3592.\n\n\nDiscussion\n\nBacteriocins are secondary metabolites in the form of proteins that act as antimicrobial compounds. The activity of bacteriocin-crude extracts of bacteria from tiger shrimp before precipitation in ammonium sulphate [(NH4)2SO4] was as presented in Table 1, and after precipitation with the same salt was shown in Table 2. Protein precipitation using 80% ammonium sulphate salt was a partial protein purification technique (partial purification) that is frequently applied as it has high solubility and is easy to obtain. Bacteriocins can be concentrated through the application of methods of salting out using ammonium sulfate as a natural protein15. Salting out is a mechanism of protein precipitation as a result of reduction in solvent molecules required to dissolve the protein. This occurs due to the increase of salt concentration which can decrease protein solubility. When ammonium sulphate is added to the protein solution, the salt ions of ammonium sulphate will attract water molecules away from protein. This is due to the competition between the binding of ions of ammonium sulphate and water which causes the protein precipitates16.\n\nBacteriocin inhibitory activity against bacterial indicators was indicated as AU (Activity unit). One AU/mL was inhibition area per unit volume of bacteriocin samples tested (mm2/mL). The method used was similar to the antibacterial assay method using agar diffusion method and three Gram-negative bacteria as pathogenic indicators (V.alginolyticus, A. hydrophila, and P. stutzeri).\n\nThe bacteriocins-crude extract before being precipitated in ammonium sulphate showed inhibitory activity against three different indicators. The activity of bacteriocin-crude extract of bacterial isolate H4 from tiger shrimp was the highest (Table 1) against V. alginolyticus (117.00 ± 66.13 mm2/mL), A. hydrophila (319,91 ± 101.03 mm2/mL and P. stutzeri (178.79 ± 61.84 mm2/mL). After precipitation in ammonium sulphate, bacteriocinactivities (Table 2) increased against V. alginolyticus (610.28 ± 257.32 mm2/mL), A. hydrophila (872.93 ± 170.02 mm2/mL), and P. stutzeri (887.10 ± 169.08 mm2/mL). The H4 isolate showed the greatest inhibitory activity due to its high inhibitory activity against the three bacterial indicators. The better the bacteria inhibit pathogenic bacteria, the better the ability to produce bacteriocins.\n\nActivities of the bacteriocins-crude extract of bacteria from prawns before and after the salt precipitation in ammonium sulphate were were shown in Table 3 and Table 4, respectively. Similar to the inhibitory activity of probiotics bacteria from tiger shrimp, all probiotic bacteria from prawns produced bacteriocins which indicated the active role in inhibiting pathogenic bacteria.\n\nNote: W = Bacterial Code. Superscript of the same letters indicates no significant differences in the level of 5%. Values were average of triplicate samples ± standard deviation.\n\nNote: W Bacterial Code. Superscript of the same letters indicates no significant differences in the level of 5%. Values were average of triplicate samples ± standard deviation.\n\nThe average activity of bacteriocins-crude extract from bacteria W2 before salt-precipitation in ammonium sulphate (Table 4) against V. alginolyticus, A. hydrophila and P. stutzeri were 345.907 ± 306.160 mm2/mL, 455.022 ± 169.591 mm2/mL and 412.735 ± 209.537 mm2/mL, respectively. After the salt-precipitation in ammonium sulfate, the bacteriocin activities increase against V. alginolyticus, A. hydrophila and P. stutzeri, which were 1043.228 ± 357.102 mm2/mL, 1113.914 ± 423.026 mm2/mL and 1466, 127.626 ± 962 mm2/mL, respectively. Activities of bacteriocins produced by bacteria W2 demonstrated the greatest potential to inhibit all three pathogenic indicator bacteria. Ponce et al.,17 found that bacteriocins of Lactococcuslactis have inhibitory activity against L. monocytogenes which was 83.33 mm2/mL. This value was lower than the bacteriocin activity of bacteria W2 (1043.228 mm2/mL) against V. alginolyticus. This is likely due to the inhibition ability of bacteria W2 being closely related to the bacterial cell growth. The better the cell growth, the number of cells increases, and this will further increase the production of bacteriocins.\n\nAntimicrobial compounds such as bacteriocins are proteins produced as secondary metabolites. The production of secondary metabolites is encoded by DNA-containing genes. Formation of secondary metabolites was affected by several conditions, among which are limitations of available nutrients in the media, the addition of inducer compounds and decrease in the growth rate18.\n\nComparing the activity of bacteriocins produced by both bacteria (H5 and W2), probiotic bacteria W2 from prawns had the highest activity against the three bacterial indicators. The significant difference (P> 0.05) in the inhibition of the three bacterial indicators showed that the active protein compounds (bacteriocins) were also different19. The sensitivity of bacteria to bacteriocins is determined by the specific characteristics possessed by each bacteria. Thus, inhibition mechanism of bacteriocins against indicator bacteria depends on the specific receptors possessed by the bacteria.\n\nThe high inhibitory activity caused by bacterial bacteriocins is closely related to the bacterial indicators which belong to Gram-negative bacteria as shown in Table 5. Bacteriocins produced by Gram-positive bacteria have low inhibitory activity, or none at all, against Gram-negative bacteria20. For example, the activity generated by Lactobacillus lactis against E. coliis very low21. Bacteriocins can inhibit or kill pathogenic bacteria when the bacteria have a close relationship19. Such as bacteriocins produced by Lactobacillus sp. SCG 1223 has a high inhibitory activity against L. monocytogenes (1648.500 mm2/mL)22. High activity was also obtained from bacteriocins produced by Lactobacillus lactis against Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus and Enterococcus faecalis21.\n\nBacteriocins analysis was performed by HPLC system after purified with salt ammonium sulfate [(NH4) 2SO4]. This analysis used a wavelength of 210 nm and 250 nm because, at these wavelengths, bacteriocins produced by Lactococcus mesentoroides show high purity13. The mobile phase used was methanol while the steady phase was silica gel. Bacteriocins analysis results were shown as in Figure 1 and Figure 2. Bacteriocins produced by all the probiotic bacteria after analysis cannot be considered as the pure product because many peaks appear in the chromatogram analysis. This proves that the new protein sample was just partially purified using ammonium sulfate salt [(NH4) 2SO4]. Bacteriocins were protein compounds that the separation should be done with a variety of purification methods as has been done by Smaoui et al.,23, bacteriocins with code BacTN635 initially purified by ammonium sulfate [(NH4) 2SO4], followed by gel filtration chromatography, HPLC, and SDS-PAGE electrophoresis. Nisin isolated from Lactococcus lactis was also purified by various purification methods, which was initially precipitated with ammonium sulphate salt [(NH4)2SO4], and was continued by purification using various chromatographic methods8,24. Nissin was the first type bacteriocin allowed as biopreservation on food5.\n\nBacteriocins were produced by probiotic bacteria H4 from tiger shrimp and probiotic bacteria W2 from prawns which were purified by gel filtration chromatography using Sephadex LH-20. The results of the sample separation were characterized by FTIR spectroscopy to identify functional groups contained in the functional group of the bacteriocins based on the literature of various types of bacteriocins by Sakhamuri et al.,24 and Selvendran and Babu25 as shown in Table 6.\n\nThe infrared spectrum of bacteriocins H4 at wave numbers (cm-1) of 971, 977, 978, 979, and 988 indicated the presence of bending vibration of amide group (O= oxygen, C= carbon, N= nitrogen), while the wave numbers (cm-1) of 1082, 1089 and 1079 indicated a bond phosphodiester (P = O), and the wave number (cm-1) of 1647, 1648, 1649 and 1652 indicated a stretching vibration of carbonyl (C = O, amide I). The infrared spectrum bacteriocins obtained relate to specific functional groups of nisin which was similar to that reported by Sakhamuri et al.24. The wave numbers (cm-1) 3336, 3415, 3420, 3443, and 3445 indicated the presence of N-H stretching vibration. Bacteriocins produced was the result of probiotic bacteria isolated from black tiger shrimp and prawns, one of which has 99% homologous with the bacteria Bacillus sp.26.\n\n\nConclusions\n\nBacteriocins had been explored from probiotic bacteria isolated from black tiger shrimp and prawns. The bacteriocins show potential as anti-pathogens in shrimp culture or in fish farming. Bacteriocins of from H4 isolate from tiger shrimp and bacteriocins of W2 isolate from prawns were two antimicrobial compounds which had the greatest inhibitory activity against all three pathogens, Vibrio alginolyticus, Aeromonas hydrophila and Pseudomonas stutzeri. HPLC analysis of the bacteriocins produced by 18 of probiotic bacteria did not show a high degree of purity. FTIR analysis of the purified products of H4 bacteriocins showed an amide bond at a wavelength of 1652 cm-1 which indicated that the compound was a protein. Bacteriocins produced from the laboratory isolates may be useful as a food preservative for controlling microbial deterioration, enhancing the hygienic quality, and extending the self-life of fish and seafood products27.\n\n\nData availability\n\nDataset 1: Word document containing the following data tables: 10.5256/f1000research.13958.d19845828\n\nCrude extract of bacteriocins of probiotic bacteria isolated from black tiger shrimp before being precipitated in ammonium sulphate [(NH4) 2SO4].\n\nThe activities of bacteriocins-crude extract of probiotic bacteria isolated from black tiger shrimp after precipitation in ammonium sulphate [(NH4) 2SO4].\n\nThe activities of bacteriocins-crude extract of probiotic bacteria isolated from prawns before precipitation in ammonium sulphate [(NH4) 2SO4.\n\nDataset 2: The bacteriocins activities of probiotic bacteria isolated from prawns after precipitation in ammonium sulphate [(NH4)2SO4]. 10.5256/f1000research.13958.d19845929", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by the Ministry of Research, Technology and Higher Education (Ristekdikti) of the Republic of Indonesia Grant No. 486/UN.19.5.1.3/PP/2017.\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\nGrateful thanks goes to the Ministry of Technology, Research and Higher Education of Indonesia who has supported this research.\n\n\nReferences\n\nFAO/WHO: Probiotics in food: Health and nutritional properties and guidelines for evaluation. Food and Agriculture Organization of the United Nations and World Health Organisation, Córdoba, Argentina, 2001. Reference Source\n\nSanders ME: How do we know when something called “Probiotics” is really a probiotics? A guideline for consumers and health care professionals. Functional Food Reviews. 2009; 1(1): 3–12. Reference Source\n\nSharma D, Singh Saharan B: Simultaneous production of biosurfactants and bacteriocins by probiotic Lactobacillus casei MRTL3. Int J Microbiol. 2014; 2014: 1–7, 698713. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanmani P, Kumar RS, Yuvaraj N, et al.: Comparison of antimicrobial activity of probiotic bacterium Streptococcus phocae P180, Enterococcus faecium MC13 and Carnobacterium divergens against fish pathogen. World Journal of Dairy and Food Sciences. 2010; 5(2): 145–151.\n\nYang SC, Lin CH, Sung CT, et al.: Antibacterial activities of bacteriocins: application in foods and pharmaceuticals. Front Microbiol. 2014; 5: 241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKarpiński TM, Szkaradkiewicz AK: Characteristic of bacteriocines and their application. Pol J Microbiol. 2013; 62(3): 223–225. PubMed Abstract\n\nDe Vuyst L, Leroy F: Bacteriocins from lactic acid bacteria: production, purification, and food applications. J Mol Microbiol Biotechnol. 2007; 13(4): 194–199. PubMed Abstract \| Publisher Full Text\n\nFawzya YN: Preservative nisin: Application in fishery products. Squalen. 2010; 5(3): 79–85. Publisher Full Text\n\nFeliatra F, Yoswaty D, Lukystyowaty I, et al.: The potential of the isolated probiotics bacterial from giant prawns’ digestive tract (Macrobrachiumrosenbergii, de Man) with 16S rDNA sequencing technique. International Journal of Oceans and Oceanography. 2015; 9(1): 1–10.\n\nSharma N, Kapoor R, Gautam N, et al.: Purification and characterization of bacteriocin produced by Bacillus subtilis R75 isolated fermented chunks of mung bean. Food Technology Biotechnology. 2011; 49(2): 169–176. Reference Source\n\nMartins IM, Cortés JC, Muñoz J, et al.: Differential activities of three families of specific beta(1,3)glucan synthase inhibitors in wild-type and resistant strains of fission yeast. J Biol Chem. 2011; 286(5): 3484–3496. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWidhyastuti N: Purification and Characterization of Extracellular xylanase Streptomyces. sp. SKKI-8 from Sukabumi. Thesis. Institut Pertanian Bogor. 2007.\n\nMasuda Y, Ono H, Kitagawa H, et al.: Identification and characterization of Leucocyclicin Q, a novel cyclic bacteriocin produced by Leuconostoc mesenteroides TK41401. Appl Environ Microbiol. 2011; 77(22): 8164–8170. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSudjana: Design and analysis of experiments. Tarsito. Bandung. 1991.\n\nPingitore EV, Salvucci E, Sesma F, et al.: Different strategies for purification of antimicrobial peptides from lactic acid bacteria (LAB). In Vilas AM, (Ed.) Communicating Current Research and Educational Topics and Trend in Apllied Microbiology. 2007; 557–568. Reference Source\n\nBintang M: Biochemical Research Technique. Erlangga, Jakarta. 2010.\n\nPonce AG, Roura SI, Del Valle CE, et al.: Antimicrobial and antioxidant activities of edible coatings enriched with natural plant extracts: In vitro and in vivo studies. Postharvest Biol Technol. 2008; 49(2): 294–300. Publisher Full Text\n\nNofiani R, Nurbetty S, Sapar A: Antimicrobial Activities Of Methanol Extract From Unidentified Sponge Associated Bacteria In Lemukutan Island, Kalimantan Barat. Jurnal Ilmu dan Teknologi Kelautan Tropis. 2009; 1(2): 33–41. Publisher Full Text\n\nDesniar, Rusmana I, Suwanto A, et al.: Screening bacteriocins of lactic acid bacteria from bekasam. Jurnal Pengolahan Hasil Perikanan Indonesia. 2011; 14(2): 124–133. Reference Source\n\nDobson A, Cotter PD, Ross RP, et al.: Bacteriocin production: a probiotic trait? Mini Review. Appl Environ Microbiol. 2012; 78(1): 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajaram G, Manivasagan P, Thilagavathi B, et al.: Purification and Characterization of a Bacteriocin produced by Lactobacillus lactis isolated from marine environment. Adv J Food Sci Technol. 2010; 2(2): 138–144. Reference Source\n\nUsmiati S, Marwati T: Seleksi dan Optimasi Proses Produksi dari Lactobacillus sp. J Pascapanen. 2007; 4(1): 27–37. Reference Source\n\nSmaoui S, Elleuch L, Bejar W, et al.: Inhibition of fungi and gram-negative bacteria by bacteriocin BacTN635 produced by Lactobacillus plantarum sp. TN635. Appl Biochem Biotechnol. 2010; 162(4): 1132–1146. PubMed Abstract \| Publisher Full Text\n\nSakhamuri S, Boner J, Irudayaraj J, et al.: Simultaneous determination of multiple component in nisin fermentation using FTIR spectroscopy. Journal of Scientific Research and Development. 2004; 1–16. Reference Source\n\nSelvendran M, Michael BM: Studies on Novel bacteriocin like inhibitory substance (BLIS) from microalgal symbiotic Vibrio spp MMB2 and its activity against aquatic bacterial pathogens. J App Pharm Sci. 2013; 3(02): 169–175. Reference Source\n\nFeliatra F, Lukistyowati I, Yoswaty D, et al.: Phylogenetic analysis to compare populations of acid tolerant bacteria isolated from the gastrointestinal tract of two different prawn species Macrobrachium rosenbergii and Penaeus monodon. AACL Bioflux. 2016; 9(2): 360–368. Reference Source\n\nLim ES: Inhibitory effect of bacteriocin-producing lactic acid bacteria against histamine-forming bacteria isolated from Myeolchi-Jeot. Fish Aquatic Sci. 2016; 19: 42. Publisher Full Text\n\nFeli F, Muchlisin ZA, Teruna HY, et al.: Dataset 1 in: Potential of bacteriocins produced by probiotic bacteria isolated from tiger shrimp and prawnsprawn as antibacterial to Vibrio, Pseudomonas, and Aeromonas species on fish. F1000Research. 2018. Data Source\n\nFeli F, Muchlisin ZA, Teruna HY, et al.: Dataset 2 in: Potential of bacteriocins produced by probiotic bacteria isolated from tiger shrimp and prawnsprawn as antibacterial to Vibrio, Pseudomonas, and Aeromonas species on fish. F1000Research. 2018. Data Source" }	[ { "id": "32653", "date": "24 Apr 2018", "name": "Hazel Monica Matias-Perala", "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 contained important results from a research activity aimed to isolate potential probiotic bacteria which produce potent bacteriocin. Upon isolation of bacteriocins, they were tested for potential antibacterial activity against pathogenic bacteria, through the utilization of disc diffusion method/ assay. After which, the bacteria that perform the best in the disc diffusion method was chosen (one from the tiger shrimp (H) and another one from freshwater prawn (W)) was further purified and subjected to HPLC and FTIR analysis.\n\nThe Introduction section clearly stated the importance of doing this research which is the utilization of bacteriocin to help in alleviating the problems of pathogens in aquaculture particularly the fish and shrimp culture. The authors also cited recent references to indicate the timeliness of this work.\n\nThe methods presented are clear with enough details for easy replication in the future. However, few details were left out in the HPLC analysis which has to be added to the manuscript: (Please indicate in the HPLC method the information on how much (microliter) of the bacteriocin samples were injected, what is the solvent used, how many percents of solvent are added and etc.\n\nThe statistical analysis is appropriate. Although it is suggested to add up which dataset were subjected to ANOVA and DMRT.\n\nThere are few things to clear up in the results section.\n\n- Please check the results which the author considered \"significantly different\" throughout the results. Similar letters indicate no significance, therefore as an example, \"ab\", \"bc\", and \"b\" are not significantly different.\n\n- Table 1, H1 - under A. hydrophila, indicate the real value (there seemed to be a problem).\n- Table 3 was not cited in the text.\n- Why is Table 5 not in presented in the \"Results\" section, but is discussed in the \"Discussion\" section? If the data tabulated in Table 5 is from your own research, it is more appropriate to present in the results section, rather than only cite in the Discussion section.\n\nThe results are considered only partially discussed in the Discussion section. Therefore it will be best to add more appropriate justifications. Following are suggested in the discussion section for improvement:\n- Tables and figures should only be cited minimally in the discussion section since they have already been presented in the results section.\n- Explain more the implication of increasing inhibitory activity against pathogens after precipitation in ammonium sulfate [(NH4) 2SO4]. Will it help in terms of increased efficiency of bacteriocin? How about in terms of the economics?\n- It will be best to expound on the result of the inhibitory activity of the bacteriocin comparing the current results to that of those previously been used. In the methodology, there was a mention of positive and negative control but the result was not used in the argument on how efficient the currently isolated and purified bacteriocin in this study.\n- There should also be a clear explanation of the reason why H4 and W2 were chosen among other isolates. In here the statistical results will become handy in explaining why.\n- Include more explanation on the FTIR results for the two chosen isolate which are potentially good source of potent bacteriocin. What were the implications of the FTIR results having the specific wave numbers as in the current results? Do they mean more potent? better than others?\n\nAs for the conclusion section: It is not advisable to cite any reference in the conclusion section since conclusion should contain the authors' own interpretation of the results collected from the study.\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": [] }, { "id": "37670", "date": "07 Sep 2018", "name": "Sriram Seshadri", "expertise": [ "Reviewer Expertise Metabolic disorder", "gut microbiota", "liver inflammation", "probiotic characterization" ], "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\nLot of words are repeating in the same sentence in the abstract.\n\nHow do you produce bacteriocin by centrifugation?\n\nThe species name of shrimp and prawn is missing in the whole test.\n\nHow was the isolates obtained? What was the sample used?\n\nHow was the health status of the shrimp and prawn confirmed?\n\nCharacterization of probiotic features are completely missing.\n\nHave the authors confirmed whether the isolates by itself had antibacterial potentials?\n\nIf so, have they determined the mode of action before moving on to bacteriocin isolation and purification?\n\nHow do the authors justify selection of only Amoxicillin as the test antobiotics?\n\nThere are no citations provided for Gel filtration chomatography.\n\nThere is no mention of standard probiotic strain which are known to produce bacteriocins. How did the authors compare the bacteriocin production and its efficacy?\n\nThe chemical formula of Ammonium Sulphate is (NH4)2SO4 not (NH4)2SO4. It has to be rectified throughout the text.\n\nBacteriocin inhibitory activity is indicated as AU. AU does not stand for Activity Unit by Arbitrary Unit.\n\nDiscussion is very poorly written. It has to be re-written and there is no mention of any papers on bacteriocins isolated from prawn/shrimps/fish sources.\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? 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": [] }, { "id": "38186", "date": "20 Sep 2018", "name": "Tati Nurhayati", "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 manuscript describes the isolation of bacteriocin derived from probiotic bacteria isolated from shrimp and lobster. This compound is tested against pathogenic bacteria. However, there are some improvement notes in some parts.\nAbstract\nThe research objective in the abstract has not been clearly written. Are bacteriocins obtained from shrimp or from bacteria that live on shrimp? There is a typo in the method section in the abstract. Bacteriiocin was written 2 times. In the abstract: the sentence Bacteriocin activity against is written 2 times. The results of chromatographic column analysis and HPLC have not been written in the results section of the abstract.\n\nMethodology\nAssay of bacteriocin activity, need to be re-examined. Need to be written clearly, how many bacteriocins are inserted into each well? In order to be able to track how to calculate the inhibition per ml. Unit diameter should be mm NOT mm2. In methodology purification methods, the use of ammonium sulfate, only one concentration is 80%. What is the basis? If there have been previous studies, it is necessary to mention here referring to whose research? Use of the term the purification of proteins with ammonium sulfate is inappropriate. There is a discrepancy in the designation term. In the abstract, it is stated that purification is done by chromatography column. But in the methodology, protein purification is carried out with ammonium sulfate. In the methodology, there is step gel filtration. What is the use of gel filtration for? For purification only, or for specific analysis. Need to write a clear title in this method. In the methodology, analysis with HPLC is used to analyze what. Need to be written in the methodology.\n\nDiscussion\nEspecially for the discussion of gel filtration, only the stages of the method are explained. The method stage should be written on the methodology not in the results and discussion. In sub discussion, it is better not to repeat mentioning the actual data already in the results section. In this sub-chapter, what should be written is the discussion of the data in the results section.\n\nConclusion\nThe last sentence from the conclusion is incorrect, because the researchers did not test bacteriocin activity against spoilage bacteria but against pathogenic bacteria.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "38187", "date": "10 Oct 2018", "name": "Eddy Afrianto", "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 the field of fisheries, the attack of pathogenic microbes and the use of formaldehyde are some of the main issues. The use of formalin as a food preservative has reached dangerous stage, but its more effective substitute compounds have not been successfully developed. Bacteriocin is a compound that has great potential to be developed as a prevent attacks of pathogenic bacteria and preservative inhibitor to replace formaldehyde.\n\nBacteriocin is a secondary metabolite product from lactic acid bacteria (BAL). These bacteria are often found in various places, including the digestive tract of fish and shrimp. There have been many efforts made to get bacteriocin-producing microbes. Increased mass production of bacteriocin is very helpful for fish cultivation activities in preventing pathogenic microbial attack and processing of fishery products as preservatives replacing formalin.\n\nThis research is the initial stage of the effort to develop bacteriocin as a deterrent to the activity of pathogenic microbes and decay. Bacterial isolates that have been found need to go through several stages in order to produce bacteriocins, including isolation processes to produce pure cultures and optimal bacteriocin separation technology from microbial culture media.\nIn the abstract several words are repeated, reducing the use efficiency of the word.\n\nThe reasons put forward to carry out this research are quite relevant. It is hoped that bacteriocin can be found that can overcome the problem of pathogens that attack fish and shrimp.\n\nThe method used has been presented in sufficient detail so as to facilitate future replication. Some parts still need to be detailed further, for example need further explanation on how to get bacteriocins using centrifugation, how many bacteriocin samples are used in the HPLC method, what and how many solvents are used?\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/7-415
https://f1000research.com/articles/7-411/v1	29 Mar 18	{ "type": "Research Article", "title": "Early storybook reading and childhood development: A cross-sectional study in Iran", "authors": [ "Firoozeh Sajedi", "Elham Habibi", "Nikta Hatamizadeh", "Soheila Shahshahanipour", "Hosein Malek Afzali", "Firoozeh Sajedi", "Nikta Hatamizadeh", "Soheila Shahshahanipour", "Hosein Malek Afzali" ], "abstract": "Background: Development is a process that continues from childhood to death, and most developmental changes occur during childhood. UNICEF introduced early storybook-reading (ESR) and storytelling as part of child care indicators. The aim of this study was to investigate the status of book-reading to children and its relationship with early childhood development in Iran. Methods: This is a descriptive-analytic study conducted in Tehran April-May 2017. In total, 272 mothers of children aged 3-30 months, who were referred to health centers, were selected using a convenience sampling method. Exclusion criteria was scoring below the cutoff point of any developmental domains of the Ages and Stages Questionnaire (ASQ). ESR was assessed by checklist and child development was assessed by the ASQ. Data were analyzed using SPSS. Results: The mean number of children’s books owned was 10.23±8.642, and 84.75% had at least 3 books. The average book reading, storytelling and singing duration for children was 10±9.65, 11.48±11.756, and 23.88 ±17.880 min per day, respectively. Average book reading, storytelling, and singing duration was significantly greater in children 18-30 months than <17 months. There was a significant relationship between the number of books and a child's age, mother's age, family income, income satisfaction, father's employment, and parents’ education. The score of communication domain in the ASQ questionnaire was significantly related to the number of books, duration of reading and storytelling, while problem-solving had a significant relationship only with the number of books (p˂0.05). Based on linear regression, child's age, income, and mother's and father's educational level were models for predicting the number of children's books (p=0.0001 for all). Conclusions: ESR was associated with some developmental domains of communication and problem-solving in the present study. Therefore, creation of ESR culture in Iranian families as an integral part of the life of children is necessary from birth.", "keywords": [ "Book Reading", "Child Development", "Early Interventions" ], "content": "Introduction\n\nDevelopment is a complex process through which an individual acquires various capabilities to improve performance and have better adaptation to the environment. Although, this process continues from childhood to death, most developmental process occurs during the first few years of life1, during which the neural structure evolves2. Although the opportunity for early childhood interventions is short, it has significant results3, and it is believed that a better beginning of life will lead to a better future4. Therefore, providing the best beginning for children can lead to success in life, employment, higher earnings, active participation in society, increased responsibility, and reduction of crime and some chronic diseases5,6. The prevalence of developmental disorders in Iranian children is between 7 and 22.4%7 and is reported to be 14–20% in other countries8–11. However, by encouraging mothers and community participation, and timely and appropriate intervention in controlling the risk factors, the ability to promote development, health, well-being and suitability of the child can also be provided12–14.\n\nMany social factors and environmental stimuli are effective in health and early childhood development (ECD)12, which include useful books and toys that provide a healthier life in the long run for the individual. Book reading is regarded as one of the most powerful protections for mental growth and development, and psychological and social stimulation of a child at home15,16. UNICEF introduced early storybook reading (ESR) and storytelling as child care indicators16. In addition, the parent-child's common interest and joint attention in reading picture books provides the basis for social and emotional communication during childhood. Reading books to children promotes their development of speech and language, and cognitive and emotional social behavior17–19, and provides them with joy20. In addition, the infant learns how to take the books in their hands and try to turn its pages. Hence, it can promote gross and fine motor coordination and also a love for reading21. When book reading is started in early months of life, a longer and more stable effect on child development will be observed22, as reported by Senechal and LeFevre23 who found that parents should begin book reading to infants at the age of 4 months, since these children had better literacy skills at school age. It is believed that ESR promotes the development of children; Murray and Egan24 reported a positive relationship between reading books and the score of cognitive development in 9-month-old infants. Cline and Edwards stated that ESR in addition to enhancing child's development and learning skills, also creates an interest, motivation and habit of regular book reading in the individual in the future22. Therefore, it is good for all family members to participate in book reading to children, and children’s books should be made available for them at home22. Additionally, during reading, a warm and intimate interaction between adults, especially parents and the children is created, and along with playing, can create love and reading infrastructure for motivation in later years22,25. In addition to book reading, storytelling and singing also have similar benefits in children and can be done anywhere or any time22. It is believed that ESR, storytelling and singing to children are a series of activities that help in the development of speech and language, literacy and children's brain development26. The most important ESR barriers are inadequate access to children's book27, and parents' lack of awareness on the positive effects of ESR and inappropriate strategies for promoting children development27–29.\n\nIn general, few studies have been conducted on the role of children's books in the lives of children under the age of three30. Reading books to children is considered as an important activity in Western culture31, including the BookStart program in the United Kingdom, which over two decades implemented reading books in the early years of life has as an Early Interventions (EI)32. Although there have been numerous studies on ECD in Iran, reading to young children is not considered as one of the environmental stimuli affecting ECD. Accordingly, this study aimed to examine the status of book reading on Iranian children, including children's books, parents' participation in reading, storytelling and singing to children based on ECD in infants and toddlers without any developmental delay during early childhood in Tehran.\n\n\nMethods\n\nThis is a descriptive-analytic cross-sectional study that aimed to investigate the status of book reading on young children and its relationship with ECD in Tehran from April to May, 2017.\n\nThe research population consisted of all mothers with children from 3 to 30 months, who were referred to one of the public health centers in Tehran for various health-related services. For sampling, Tehran was divided into two regions, East and West, and one health center was selected from each of the two regions randomly Ershad and Shahre Ziba public health centers were selected in the East and West of Tehran, respectively. Sampling of mothers was conducted using convenience sampling method. Mothers that were referred to the health center for a routine checkup for her child, child vaccination and/or receiving other health services, were entered in the study.\n\nUsing a sample size formula1 and a 95% confidence level, and also considering the prevalence of developmental disorders, which was considered to be about 22% in Iran in accordance with previous studies7, with an increase of 10% to take account of potential attrition, 290 people were enrolled in the study, from which 272 consented to participate.\n\nThe criteria for entry in this study were parents with a child aged 3 to 30 months who, while referred to the health centers, who were willing to cooperate. At first, the aims of the study were explained to the parents once they were referred to the health center. They also provided written informed consent and completed self-report questionnaires in about 10–15 minutes. The mothers of children with developmental delay (based on ASQ) were excluded.\n\nThe assessment tools of this study were the ASQ, demographic questionnaire and a checklist for assessing the ESR status of children (Supplementary File 1). The demographic questionnaire and checklist were prepared by the research team and validated by 7 pediatricians and specialists in child development who worked in University of Social Welfare and Rehabilitation Sciences of Iran. These tools were piloted by 8 mothers that were referred to the Saadat Abad Health Center. This checklist included the number of children's books, duration of reading, storytelling and singing for children as variables.\n\nThe ASQ is used to screen infants and young children for developmental delays during the crucial first 5 years of life. This tool assess the different developmental domains (gross motor, fine motor, communication, problem-solving, and social-personal)33. For each age group, a total of 30 questions (six questions for each developmental domain) were designed and the highest score available for each question is 10; therefore for each developmental domain, the score is 60. The answer to each question included yes, sometimes and never with 10, 5 and 0 points considered for each question, respectively. This questionnaire is a standard tool that has been translated into various countries in Asia, Europe, Africa and the United States34,35, and the sensitivity and specificity of this test are 88% and 82.5%, respectively35,36. The screening of developmental delay was done by comparing the score of each area with a cut-off point37 based on the age of Iranian children. According to a study by Sajedi et al.38 the Cronbach's alpha obtained was 0.79 and, the validity, reliability, and ability of the test for determination of evolutionary deficits were 0.84, 0.94 and 96%, respectively37. As it is possible that parents may rate their children more favorably, and the ASQ is a self-report questionnaire, the mothers were given adequate explanations of each question and answer before completing the questionnaires in order to reduce reporting bias.\n\nDue to the nature of the present study, demographic data, ESR status for children, and ASQ questionnaire scores were standardized in terms of mean and standard deviation. Categorical data were summarised using absolute values (percentage). After determining the data normality of each variable, Chi-square, independent t-test, and correlation coefficients Spearman’s test were used. Considering the fact that the data gathering tools (i.e. the questionnaire) was a self-reported one, despite of all counter measures, having missing data was inevitable. Assessment of the impacts of such missing data, was made by Univariate T-Test. The results revealed that the missing data were completely at random39. In order to predict the situation of ECD, independent variables were included in the linear regression model. Data were analyzed by SPSS software version 22 at a significant level of p<0.05.\n\nThe present research was approved by the University of Social Welfare and Rehabilitation Sciences (ethics code IR.USWR.REC.1395.77). This study observed ethical standards, and after sampling, in the case of results from the ASQ showing probability of developmental delay for certain children, this was discussed with pediatricians who were in contact with parents.\n\n\nResults\n\nThe number of mothers included in the study was 272. Most of the children (86.86%) were born by cesarean section method, 50.9% were girls, and 21.5% were also cared for by grandparents in addition to their parents. As shown in Table 1, the mean age of mothers and fathers was 31.72 ± 4.450 and 35.5 ± 5.149 years, respectively. 68.4% of mothers were housewives and 5.5% of fathers were unemployed. The average income of families was 31,150,000 Rials, 54.8% of whom were satisfied with their income. 73% of children did not have any siblings.\n\nOn average, every child had 10.2±8.642 books. Among the children, 30 (11.20%) did not have any books and 228 (84.75%) had at least 3 books. Only five children (1.83%) had more than 37 books each. The average daily reading time for children was 9.65 ± 10, while for half of them, nobody read 5–15 min to them a day, and 27.6% of them were never read to. The average storytelling time for children was 11.48 ± 11.756 min per day, while 25.7% of parents had never told the children a story. The average duration of singing for children was 23.88 ± 17.880 min per day, but for 11.4% of them, nobody has ever sung.\n\nAccording to the results in Table 2, the number of children's books owned, duration of ESR, storytelling and singing in children was significantly different between children aged 3 to 17 months and those aged 18–30 months (p=0.0001 for all). There was a significant relationship between the number of children’s book owned with duration of ESR, storytelling and singing (p<0.0001). In addition, there was a significant association between the number of children's book owned and children's age (p<0.0001), mother's age (p=0.021), family income (p=0.009), income satisfaction (p=0.026), father's occupation (p=0.04), and mother's and father's educational level (p<0.0001). ESR duration for children was significantly correlated with the age of the child, mother and father (p<0.0001). Singing duration was significantly correlated with only the mother's educational level (p=0.011).\n\nThe correlation between ESR variables is shown in Table 3. From the viewpoint of development, the scores of communication and problem-solving from the ASQ questionnaire showed a significant relationship with the number of children's books owned (p<0.0001 and p=0.016). Also, there was a significant correlation between the score of communication and duration of ESR and storytelling (p=0.002 and p=0.028). There was no significant relationship between variables and duration of singing (P>0.05 for all variables). Based on linear regression, children's age, income, and mother's and father's educational level can be models for predicting the number of children's books owned (p<0.0001 for all).\n\n*Correlation is significant at the level 0.05 (2-Tailed).\n\n\nDiscussion\n\nIn the present study, the average number of children’s books owned was 10.23±8.642, which increased significantly in infants aged 18 to 30 months as compared to infants less than 17 months old (p=0.0001). In other studies, this number was reported to be 2.6± 3.600 to 7.9± 10.9 for children aged 6 to 18 months, indicating an increase in the number of children's books owned based on the age of the children, and it is believed that during early infancy, playing is considered more important than book reading30. A total of 84.75% of the children had at least 3 children’s books; this was reported to be 97% in Ukraine and 3% in Laos16. According to the results of the current study, more than 68% of the parents had a university education, although in Shahshahani et al.’s40 only 34% of parents had a university education. In the present study, there was a significant relationship between the number of books and the duration of ESR with parental education, while in this case Boyle and colleagues believed that educated mothers used better medical education recommendations41, while fathers with university education can provide a more sustainable financial and social environment for their children21. In addition, mother's level of education plays a facilitating role in the book reading of children and ECD21. According to some similar studies, educated parents also read more books to their children42. However, in the study by Tomopoulos et al.30, maternal education was not associated with ESR. The mean ESR time in the current study for children aged less than 17 months and from 18 to 36 months was 7.42±8.571 and 13.04±11.058 minutes per day, while in the study of 30, the mean for the ages of 6 and 18 months was 2.1±2.300 and 3.5±2.800 days per week, respectively. Although in the present study, father's employment was significantly correlated with the number of children's books owned; there was no significant relationship between father's occupation and ESR, which is similar to result presented in 30.\n\nGiven the average number of books in this study, for 61.4% of children aged 0–17 months, book reading was begun at the time of sampling; this statistic increased to 86.5% in children aged 18 to 30, while based on the results of Duursma21, book reading was done daily or weekly for 77% of the children who were 14 and 24-months-old, 52% of children aged 4 to 35 months43 and 50% of children aged 0 to 36 months27. According to Senechal and LeFevre23, the number of picture books in a home has a strong relationship with the children's receptive and expressive language. Therefore, parents should be advised to read books to their children at the earliest opportunity, from birth, because it can be effective in their development5. Meanwhile, in the current study, 28.6% of the children had never been read to, while in other studies, this statistic was 4% in the UK27 and 6–23% in the USA21, 43.\n\nAccording to the present findings, family income showed a significant relationship with the number of books owned and duration of ESR. In this regard, Karrass and his colleagues44 also found in 2013 that mothers of higher income families had a greater tendency for ESR for their 8-month-old infants. Additionally, according to a UNICEF report16 in 2012, children from poor families had fewer children’s books at home.\n\nIn the present study, 62.5% and 90.4% of children aged 0–17 and 18 to 30 months were told stories. Also, 84.6% and 94.24% of children aged 0–17 months and 18 to 30 months were sung to, indicating an increase in these stimuli with the age of the child. In confirmation, other researchers also believe that storytelling and singing to children helps in speech evolution, language and literacy, as well as the evolution of the baby's brain45.\n\nHaving a book and reading it at home will provide more opportunities for improving skills46. According to the results of the current study, there was a positive correlation between the number of children's books owned and problem-solving scores. In confirmation of this result, by using ASQ, Murray and Egan’s research also found that reading books to infants had a positive impact on problem-solving and communication issues11. In this regard, Duursma21 also proposed the ability to predict 24-month cognitive skills score by Bayley MDI based on the time spent in reading to a child. Also, loud book reading had a significant effect on the cognitive development of premature infants after two years47. In the present study, there was a positive correlation between the number of children's books owned, the duration of ESR and storytelling for children and the communication score. In relation to the impact of ESR on speech-language development, results of research indicates that children use a rich vocabulary during book reading18,31 and interventions for the development of the children's language, focusing on reading books whenever possible in children's lives are required48. However, in the current research, there was no relationship between ESR and other developmental domain scores, but in another study it was reported that ESR may promote language and social communication skills49.\n\nIn this study, the important environmental stimuli (having a book at home and its number, and duration of book-reading, storytelling and singing) in children without any developmental delay in Tehran, was described, thus there was no information about ESR and its effect on children with developmental delay. Therefore, it is suggested that, in addition to conducting more extensive studies in the country, the quality and style of book reading for children should be addressed in the future in Iran.\n\n\nConclusion\n\nAccording to the results of this study, ESR is considered as an early environmental stimulus and interventions in domains of communication and problem-solving in children. Therefore, the establishment of ESR culture among families and individuals in the Iranian community as a recreational and inseparable part of children's life from birth is recommended. As a result, due to the differences between culture and traditions among Iranian people, it is suggested more extensive studies be conducted on the quality, style of reading, and the views of parents and other caregivers on EI when dealing with reading books to children and its effects on ECD in Iran. The results of this study are only generalizable to Iranian children of Tehran.\n\n\nData availability\n\nDataset 1: ESR and ECD data from the East and West of Tehran. Variables are coded as follows: Birth rank: 1=The first child, 2=The second child and more; Caregivers: 1=mother, 2=father, 3=grandparents, 4=others; Age group: 0=3 to 17, 1=18 to 30; New Gestational age: 1<37 Week, 2≥37 Week; New mother educate: 1=diploma and below, 2=academic; New father educate: 1=diploma and below, 2=academic; New mother job: 1=Employed, 2=Housewife; New father job: 1=Employed, 2=Unemployed. DOI, 10.5256/f1000research.14078.d19865750\n\n\nFormula\n\n1n= (Z1–α2)2×P(1–P)d2", "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 express their special gratitude to a number of people who kindly provided their insightful and invaluable advice and support throughout all the stages of this research. This study is part of a research-based doctoral dissertation in Pediatric Neurorehabilitation Research Center, University of Social Welfare and Rehabilitation Sciences, Iran. The authors also acknowledge all the staff in this center and university without whose cooperation, this process could not have been fulfilled.\n\n\nSupplementary material\n\nSupplementary File 1: Survey of demographic and ESR in Persian with an English translation.\n\nClick here to access the data.\n\n\nReferences\n\nBhattacharya T, Ray S, Das DK: Developmental delay among children below two years of age: cross-sectional study in a community development block of Burdwan district, West Bengal. Int J Community Med Public Health. 2017; 4(5): 1762–7. Publisher Full Text\n\nO'Mahony SM, Clarke G, Dinan TG, et al.: Early-life adversity and brain development: Is the microbiome a missing piece of the puzzle? Neuroscience. 2017; 342: 37–54. 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Data Source" }	[ { "id": "32641", "date": "17 Apr 2018", "name": "Marzieh Rostami Dovom", "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 sharing the manuscript entitled “Early storybook reading and childhood development: A cross-sectional study in Iran” with me. I read the paper carefully and find it is credible and appropriate for publication and it doesn’t have any major problem. This is a new topic in Iran and the results of this research can be the base for further research on book reading for children. I think the methods and the results sections are written completely and in clear format. Only as minor comments please follow below points:\n\nThe limitations are more based on suggestions, and the main limitation of the research is not clearly stated.\n\nIt is suggested that, in the Ethical statement, the cut-off point for the developmental delay should also be mentioned.\n\nSome sentences of the article should be edited by a native editor.\n\nAdd the full form of acronym in the first time appeared in the article. for example: Ages & Stages Questionnaires (ASQ)\n\nDid you use a localized standard ASQ questionnaire?\n\nIn the data set, there are 273 subjects, but data analysis has done for 272 subjects? Is there any reason?\n\nAre there any relation between ESD, ECD, and infant sex or type of delivery? My mean is that the gender of infants or even the type of delivery (natural delivery or cesarean section) may have effect on the ESD.\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": "3601", "date": "19 Apr 2018", "name": "elham habibi", "role": "Author Response", "response": "Dear Editor/Dear RefereeMany thanks for your email and useful comments from the reviewer, which will certainly help to improve the paper.Referring to the comment #6, \"In the data set, there are 273 subjects, but data analysis has done for 272 subjects? Is there any reason?\" , we would like to clarify as below:With all due respect to the reviewer, we checked the data set (Excel file) again. The first row of the file contains the title of the variables. The number of samples is shown in column B as the code (272 people). Therefore, all samples are considered in the analysis.We will be pleased to receive and consider any comments from your esteemed referee, which we believe will certainly help to improve the current paper.Sincerely yours,E, Habibi" } ] }, { "id": "36960", "date": "13 Aug 2018", "name": "Leila Bazrafkan", "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 of the manuscript conducted a cross-sectional study to assess the state of early childhood development and early storybook reading in Iran. They reported a correlation between Early Storybook Reading (ESR) and early childhood development in communication development domain. I support the conclusion and consider ESR as an environmental stimulation that can promotes early childhood development; it also has a key role in building up the interest in book reading in the future.\n\nAfter a detailed review of the report, it could be stated that the study is sufficiently done and reported appropriately. I generally support the main conclusions and find it suitable and acceptable for indexing.\n\nRecommendations for authors:\nIt is better to clarify the limitations of the study.\n\nIt seems necessary to specify which version of ASQ was used to assess the state of Early Childhood Development.\n\nRegarding table1,\nin data file, columns AD and AE titled as “New mother educate (1=diploma and below, 2=academic)” and “New father educate (1=diploma and below, 2=academic)” were analyzed and reported but in the first column, rows seven and eight, only “Diploma” is mentioned pleas correct it to “Diploma and below”\n\nIn the first column on row explain the table's footnote that Rials is the Iranian currency\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-411
https://f1000research.com/articles/7-408/v1	28 Mar 18	{ "type": "Research Article", "title": "Evaluation of controlled type II diabetics ascending and descending a ramp surface at an imposed speed: A case-control study", "authors": [ "Martin G. Rosario", "Elizabeth Orozco", "Nairoby Babilonia", "Greisy Tellez", "Francheska Mojica", "Maryvi Gonzalez-Sola", "Flavia Bayron", "Elizabeth Orozco", "Nairoby Babilonia", "Greisy Tellez", "Francheska Mojica", "Maryvi Gonzalez-Sola", "Flavia Bayron" ], "abstract": "Background: Patients with diabetes have been shown to suffer from increased fall risk. Research shows that this risk is higher on irregular surfaces. Existing studies evaluate gait on irregular surfaces, such as stairs, asphalt, grass and stones. This study evaluates gait parameters in individuals with diabetes mellitus type II (DMII) with no history of peripheral neuropathy, while ascending and descending a ramp at an imposed speed, and compares them with healthy controls. Methods: Fifteen healthy volunteer participants and fifteen participants with DMII and no peripheral neuropathy (females and males) between the ages of 40-65 were recruited for this study. Participants walked three times at 100 bpm while ascending and descending a wooden ramp. Temporospatial and kinematic parameters were analyzed. Results: We observed minimal changes in temporospatial and kinetic parameters in people with controlled DMII with no evidence of peripheral neuropathy. Conclusion: Focusing on individuals with controlled DMII allowed us to determine if only the diagnosis of diabetes without peripheral neuropathy influenced gait parameters. Clinicians and researchers should focus their assessments on neuromuscular activation during this stage of the condition, thus preventing complications, such as abnormal gait, that increases the risk for falls.", "keywords": [ "controlled diabetes", "ramp surface", "kinematics and temporospatial parameters" ], "content": "Introduction\n\nSignificant gait and balance deficits have been found in persons with type two diabetes mellitus (DMII) compared to individuals without DMII (Brach et al., 2008). In fact, individuals with DMII are two to three times more likely to report difficulty walking a quarter of a mile, climbing stairs, or doing housework, and require the use of an assistive device for ambulation (Cavanagh et al., 1992; Crowther et al., 2007; Wallace et al., 2002). Current evidence suggests that multiple factors contribute to these gait and balance deficits, such as impaired lower extremity strength and sensation, low physical activity levels, increased age, BMI, and time since diagnosis (Brach et al., 2008; da Cruz Anjos et al., 2017).\n\nAs a result of having gait and balance deficits, Dingwell et al. found that people with diabetes demonstrated a 15% greater fall risk than healthy individuals (Dingwell et al., 2000). The risk of falling increases by 17-fold for individuals older than 65 with diabetes (Vinik et al., 2017). Persons with DMII and peripheral neuropathy (PN) will present with characteristics of postural instability (increased postural sway and poor static balance) and demonstrate a more conservative gait pattern, with temporospatial deficits such as decreased speed, step length, stride length, and single limb support time when compared to controls without DMII (Mustapa et al., 2016). These deficits can occur because of impaired proprioceptive feedback, lower extremity muscle weakness, and potential visual deficits due to elevated glucose levels that damage the retina (Mustapa et al., 2016).\n\nHowever, recent evidence suggests that similar gait and balance deficits can be found in individuals with DMII without PN (Hewston & Deshpande, 2016). In a study by Vaz and colleagues (2013) postural control and functional strength were assessed in individuals with DMII (with or without PN) compared to controls (Vaz et al., 2013). Postural control was evaluated by observing participant’s static balance in four different conditions as well as the Berg Balance Scale. Functional strength and mobility were assessed using the Five-Times-Sit-to-Stand and Timed Up & Go, respectively. Vaz and colleagues found that both DMII groups (with and without PN) demonstrated decreased postural control and functional strength compared to healthy individuals of the same age (Vaz et al., 2013). For individuals with DMII and no symptoms of PN, these impairments are most noticeable when they are exposed to challenging everyday activities like reaching, ascending stairs, or walking over unstable surfaces, which require greater postural stability. Centomo and colleagues (2007) found that participants with DMII and no PN demonstrated greater postural instability and difficulty recovering their balance after performing a forward reach test compared to age-matched healthy controls (Centomo et al., 2007).\n\nIn addition, patients with DMII also use different compensatory strategies during walking or standing (Manor et al., 2008). In a study conducted by Mueller and colleagues (1994), participants between the ages of 35–75 were instructed to walk on an elevated 6.8m walkway, using shoes that were 2.54cm tall. According to the authors, patients with diabetes used more hip strategy than the ankle strategy during ambulation. This strategy resulted in a decrease in stride length and velocity, as compared to the control group (Mueller et al., 1994). Allet et al. concluded that walking in real-life conditions revealed gait impairments in patients with DMII (Allet et al., 2009). Onodera and colleagues (2011) found that individuals with DMII between the ages 55–62 presented with a reduction in plantarflexion during the early phase of weight acceptance while ambulating stairs. This alteration impairs the mechanism of impact on absorption and load distribution when the forefoot contacts the ground. These changes can increase the risk for ulcer formation in real-life activities. Patients with DMII and PN use an adapted motor strategy to use stairs, which promotes a biomechanical deficit giving place to a decrease in range of motion (Onodera et al., 2011).\n\nVarious studies have evaluated the gait on irregular surfaces of individuals with DMII; however, to the best of our knowledge, there is not any research that focuses on gait when ascending and descending ramps in this population. Therefore, our study is aimed at answering the following: What are the temporospatial and kinematic deficits demonstrated in individuals with DMII while ascending and descending a ramp during an imposed speed?\n\nTherefore, we hypothesized that, compared with healthy individuals, (i) individuals with controlled DMII and no evidence of PN would show an alteration in temporospatial parameters (stride length, step length, cadence, single limb support and double limb support) while ascending and descending a ramp at an imposed speed; and (ii) individuals with controlled DMII and no evidence of PN would demonstrate altered kinematic parameters of the lower limb (active range of motion of hip, knee, and ankle) ascending and descending a ramp at an imposed speed. Even with controlled diabetes and the absence of PN (non-PN cDMII), we believe that imposing a velocity of 100 beats per minute will make temporospatial and kinematic alterations more evident in this population. The imposed speed (faster than normal walking speed) adds a further challenge to the ramp surface walking. Therefore, by identifying the biomechanical properties in patients with DMII while ascending and descending this sloped surface, will enable better comprehension of the underlying mechanisms involved in this task; therefore allowing us to develop interventions that strive to prevent falls in this population.\n\n\nMethods\n\nThe study protocol was approved by the Institutional Review Board of the University of Puerto Rico Medical Sciences Campus (Protocol A2540413). Each participant read and signed an informed consent document after being informed of all the risks, their rights, and potential discomforts they could encounter while participating in this gait study. The study was conducted at a Physical Therapy Laboratory that is affiliated with the School of Health Professions at the University of Puerto Rico Medical Sciences Campus.\n\nFifteen non-PN cDMII adults (eight men and seven women; age=57.7± 5.12 years, height 167.1±4.3 cm, mass 80.1±34.3kg) formed the diabetic group (8.0±5.8 years with the diagnosis of DMII), and fifteen healthy adults (seven men and eight women, age=54.4± 6.28 years, height 164.8±3.9cm, mass 73.6± 28.9 kg) made the control group (CG) (for participant enrollment see Figure 1). Patients with type two diabetes and healthy volunteers were recruited homogeneously as per sex, age, body mass index, and level of physical activity (Table 1). Diabetics were classified as controlled diabetes which according to the American Diabetes Association is defined as individuals with a glycosylated hemoglobin level of 7.5% or less. This sample size takes into consideration the primary variables (step/stride length, cadence, the velocity of ambulation and AROM), the highly-instrumented nature and the novelty of this study. The sample size for this study is comparable to other studies that involve similar variables and population characteristics (Dingwell et al., 2000; Mueller et al., 1994; Petrofsky et al., 2005).\n\nResults of Student’s t-test performed between the two sample groups. Significance threshold = P ≤ 0.05; significant P= threshold value; non-significant P=calculated value; NA=not applicable.\n\nIndividuals with non-PN controlled DMII (cDMII) and healthy participants were recruited through flyers that were placed around the University of Puerto Rico, Medical Sciences Campus. The inclusion criteria for individuals without diabetes (control group; CG): female or male, 40 to 65 years old, and can ambulate without an assistive device. The exclusion criteria were: BMI>30, severe balance problems, current ulcer(s) or history of ulcers, the absence of sensation in the lower extremities, amputations, cardiopulmonary disease, back or lower extremity pain, disease or surgery in low back in the last year, pregnant women, or lower extremity surgery.\n\nThe inclusion criteria for the non-PN cDMII group: type II diabetes diagnosed by a physician, female or male, age between 40–65 years old. The exclusion criteria for the diabetic group: BMI>30, severe balance problem, current ulcer(s) or history of ulcers, absence of sensation in lower extremities, amputations, lower extremity surgery, cardiopulmonary disease, pregnant women, back or lower extremity pain, disease or surgery in low back within the last year, or glycosylated hemoglobin higher than 7%.\n\nIn addition to the inclusion and exclusion criteria above, all non-PN cDMII and CG participants were further screened using the AHA/ACSM Health/Fitness Participation pre-screening questionnaire. This tool assessed medical history pertinent to our study and to determine the overall health status and exercise capabilities of each participant before testing (Balady et al., 1998). To ensure a more homogeneous non-PN cDMII and CG samples, two additional steps were taken:\n\nBody mass index (BMI): BMI is the ratio of body mass (kg) divided by the square of body height (meters). Participants with BMI of 18.5 or below (underweight) and 30.0 and above (obese) were excluded from the study.\n\nStandard anthropometric measurements: The height (cm) and mass (kg) of each participant were measured with a stadiometer and scale, respectively. These measurements are essential for accurate motion tracking by the three-dimensional motion analysis system defined below.\n\nTo ensure that participants could safely partake in this study and meet the criteria, participants were further screened according to the following five clinical criteria.\n\nSensibility assessment: Any participant who was unable to detect the 10 g applied force of a 5.07 Semmes-Weinstein monofilament in more than two areas on the plantar aspect of the foot was considered to have PN (i.e., loss of protective sensation and deep pressure sensation) (Bell-Krotoski et al., 1993; Thomson et al., 2008) and excluded from the study.\n\nOxygen saturation: This noninvasive method of measuring oxygen saturation with a meter (ChoiceMMed OxyWatch C20 Fingertip Pulse Oximeter #TM66018) attached to the index finger of the left hand and the second toe of the left foot was used to define the basic respiratory function. Participants with less than 90% oxygen saturation at rest were excluded from the study.\n\nSit-to-Stand Test: This test was used to determine if the participant had sufficient lower extremity strength to complete the tasks with minimal fall risk. Each participant was required to successfully sit and stand five times in 15 seconds or less (Whitney et al., 2005). Participants who were unable to meet this standard were excluded from the study.\n\nStep (Tecumseh) Test: To test for cardiorespiratory fitness, the participants were required to ascend and descend a stair step (height: 20.32 cm) for two minutes at a rate of 96 steps/minute. Heart rate, blood pressure, and oxygen saturation were measured before and after the test. Those participants who were unable to complete the task were excluded from the study.\n\nScreening Questions (Supplementary File 1)(non-PN cDMII patients): This tool is a brief questionnaire that is used to obtain the medical history of each participant. Questions for the non-PN cDMII included the extent of their diabetes, their HbA1c values within the last three months, and the medications they have been prescribed.\n\nFor testing, the participants were instructed to wear shorts and T-shirt and no shoes or socks. Fifteen retro-reflective markers were placed on each participant based on the plug-in-gait model at anatomical landmarks on the participant’s lower limbs and pelvis (Figure 2) to define the body segments of interest to the study. The instrument used in this investigation during walking for kinematic analysis were captured with a six-camera, three-dimensional, motion analysis system (Vicon Motion System, Denver, CO, USA) recording at 120 Hz. The Vicon System measures spatiotemporal parameters and hip, knee and ankle active range of motion (AROM) during gait. Prior to each data collection session, the equipment was calibrated according to manufacturer instructions. After space calibration, a static trial with the participant standing in a T-position was performed to align joint centers coordinates to the laboratory space. Data was recorded at 120 Hz with six infrared cameras. A member of the research team demonstrated the task to the participant. Each participant had the opportunity to practice the task three times with rest intervals of at least one minute.\n\nThe retroflective markers were placed on different anatomical locations based off the plug-in-gait model.\n\nParticipants stood in front of a taped line on the floor. Each participant was instructed to walk 2.44 meters (8 feet) to the wooden ramp, ascend it, walk to the end of the leveled platform, make a U-turn, descend the ramp, and walk back to the starting point. The timer began immediately after the participant was instructed to “go” and stopped as soon as the participant crossed the line on the floor with both feet. The participant walked on this surface three times at 100 beats per minute (bpm). A metronome was set to 100bpm and participants were asked to walk in sync with the sound of the beat, thus ensuring that participants ascended and descended the ramp at a velocity of 100 bpm.\n\nStatistical analysis was performed using SPSS version 20 software package for Windows. Significant difference for each temporospatial and kinematic variables was evaluated using MANOVA. A p-value ≤0.05 was considered statistically significant. Results are expressed as the mean ± standard deviation (SD). As the participant ascended and descended the ramp at 100bpm, AROM measurements (hip, knee, and ankle) were taken at the exact moment when the foot made the change from a smooth surface to a ramp surface, at the precise moment of heel strike, which we define as maximum dorsiflexion. Also, toe off was described as maximum plantar flexion while ascending and (after the turn around) descending just before heel strike at the smooth surface. Therefore, all kinematic data (hip, knee, and ankle) was collected when the participant was at maximum dorsiflexion and plantar flexion in a sagittal plane (flex/ext). Temporospatial data was collected and analyzed the entire length of the walkway. A t-test was performed to determine the differences for each variable between the control group and non-PN DMII group (Table 1).\n\n\nResults\n\nTable 1 shows demographic and clinical variables (mean ± standard deviation), along with years of diabetes diagnosis and percent of glycosylated hemoglobin in diabetic participants. No significant difference was found in the kinematic parameters (AROM) for hip, knee, and ankle (Table 2). During the ascending of a ramp at a speed of 100 bpm, the non-PN DMII group showed a slight increase in AROM of the joints of the hip, knee, and ankle. While descending the ramp at a speed of 100 bpm, non-PN DMII participants showed a minimal decrease in the AROM of the hip, and minimal increase in AROM with knee flexion and plantar flexion, which was not significantly different from the CG.\n\nResults of analysis (MANOVA) performed between the two sample groups: controlled diabetic group without peripheral neuropathy (non-PN cDMII) and healthy non-diabetic control group (CG). Significance threshold = P < 0.05; significant P= threshold value; non-significant P=calculated value.\n\nThe temporospatial variables considered in this study were cadence, gait speed, single/double limb support, step length and stride length. The temporal parameters are cadence, gait speed, single limb support and double limb support, and the spatial parameters are step length and stride length.\n\nTable 3 shows the temporospatial parameters results with their standard deviations values. The CG and DMII participants were comparable in all variables.\n\nResults of analysis (MANOVA) was performed between the two sample groups: controlled diabetic group without peripheral neuropathy (non-PN cDMII) and healthy non-diabetic control group (CG). Significance threshold = P < 0.05; significant P= threshold value; non-significant P=calculated value.\n\n\nDiscussion\n\nThe purpose of this study was to analyze kinematics (hip, knee, and ankle) and temporospatial gait parameters while ascending and descending a ramp at a velocity of 100 bpm in people with non-PN DMII group compared to healthy controls.\n\nWe hypothesized that people with controlled diabetes and no history of PN would show significant differences in temporospatial parameters and kinematics while ascending and descending the ramp. However, our study rejects this hypothesis. Results demonstrated that the non-PN DMII group did not show significant differences in AROM of hip, knee, and ankle and any of the temporospatial parameters compared with the CG.\n\nIn our study, we did not find any differences between groups. This could be because our participants had no history of neuropathy and the testing surface was not challenging enough to detect significant deviations in kinematic or temporospatial parameters. Similar to our study, Mueller et al. (1994) found that while walking on an elevated walkway, the PN-DMII group demonstrated a decrease in dorsiflexion and plantar flexion AROM, but the reduction was not significant. However, Onodera et al. (2011) found a significant decrease in dorsiflexion and plantar flexion in their participants with diabetes and PN when they ascend stairs and a significant decrease of plantar flexion while descending stairs.\n\nIn our study, no significant difference was found in the speed of ambulation between the two groups while the ascending and descending the ramp. This outcome was expected due to the protocol that called for an imposed speed of 100bpm and the controlled glucose levels in our participants. Meanwhile, Dingwell et al. (2000) and Chiles et al. (2014) found that individuals with DMII and PN have a significant decrease in their speed of ambulation on level surfaces. In addition, Petrofsky et al. (2005) found the same in persons with DMII with no evidence of PN. Both agreed that individuals with DMII with and without neuropathy demonstrated a reduction in speed as an adaptive mechanism to feel safer while walking on smooth surfaces. Also, Allet and colleagues (2009) found that participants with DMII and DMII/PN demonstrated a significant decrease in their speed of ambulation when changing from asphalt to stones and from grass to stones, to feel safer during the ambulation.\n\nAs noted, in the early stage of controlled DMII, speed of ambulation is not significantly affected, as the reduction in speed occurs in the later stages. In our study, the most affected variable was step length, where there was a decrease in the non-PN DMII group; however, this was not significant. The stride length was shorter in the non-PN DMII group compared with the CG. In the studies of Dingwell et al. (2000), Mueller et al. (1994), and Camargo et al. (2015) found that individuals with DMII/PN showed a significant decrease in the stride length compared to the control group.\n\nFinally, we found that the non-PN DMII group showed a minimal decrease in cadence during ramp ascent and descent. In comparison, Mueller et al. (1994) and Courtemanche et al. (1996), found that individuals with DMII/PN showed a decrease in cadence (not significant) during ambulation on an elevated walkway compared to the control group. In the study of Allet and colleagues (2009) both the control group and the groups with DMII (with and without PN) showed a significant decrease in cadence when changing from one surface to another.\n\n\nConclusion\n\nThe variable that was most affected while ascending and descending the ramp in participants with controlled DMII (≤ 7% glycosylated hemoglobin) was step length. We observed minimal changes in temporospatial and kinetic parameters in people with controlled DMII with no evidence of peripheral neuropathy. Focusing on individuals with controlled DMII allowed us to determine if controlled diabetes with no history of PN influenced gait parameters, most specifically while ascending and descending a ramp. Clinicians should emphasize their assessments in these areas to prevent complications, such as gait abnormalities, that can increase the risk for falls.\n\nFor future studies, we suggest measurement of lower extremity muscular activation and different degrees of slopes, making the ramp surface more challenging.\n\n\nData availability\n\nDataset 1: Ramp ascending and descending data for the non- peripheral neuropathy controlled type 2 diabetes and control groups. DOI, 10.5256/f1000research.14401.d199072 (Rosario et al., 2018).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis article was published with support from Texas Woman’s University Libraries’ Open Access Fund.\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: Diabetes assessment screening questions.\n\nClick here to access the data.\n\n\nReferences\n\nAllet L, Armand S, de Bie RA, et al.: Gait alterations of diabetic patients while walking on different surfaces. Gait Posture. 2009; 29(3): 488–493. 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. 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PubMed Abstract\n\nMustapa A, Justine M, Mohd Mustafah N, et al.: Postural Control and Gait Performance in the Diabetic Peripheral Neuropathy: A Systematic Review. Biomed Res Int. 2016; 2016: 9305025. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Diabetes Education Program: The facts about diabetes: a leading cause of death in the U. S. U.S. Department of Health and Human Services, National Institutes of Health. Reference Source\n\nNational Diabetes Education Program: The diabetes epidemic among Hispanic/Latinos. U.S. Department of Health and Human Services, National Institutes of Health. Reference Source\n\nNational Diabetes Education Program: Los hispanos/latinos y la diabetes. U.S. Department of Health and Human Services, National Institutes of Health. Reference Source\n\nNational Diabetes Statistics: Como prevenir los problemas de la diabetes: mantenga sano el sistema nervioso. National Diabetes Information Clearinghouse. Reference Source\n\nOnodera AN, Gomes AA, Pripas D, et al.: Lower limb electromyography and kinematics of neuropathic diabetic patients during real-life activities: stair negotiation. Muscle Nerve. 2011; 44(2): 269–277. PubMed Abstract \| Publisher Full Text\n\nPetrofsky J, Lee S, Bweir S: Gait characteristics in people with type 2 diabetes mellitus. Eur J Appl Physiol. 2005; 93(5–6): 640–647. PubMed Abstract \| Publisher Full Text\n\nRosario MG, Orozco E, Babilonia N, et al.: Dataset 1 in: Evaluation of controlled type II diabetics ascending and descending a ramp surface at an imposed speed: A case-control study. F1000Research. 2018. Data Source\n\nThomson MP, Potter J, Finch PM, et al.: Threshold for detection of diabetic peripheral sensory neuropathy using a range of research grade monofilaments in persons with Type 2 diabetes mellitus. J Foot Ankle Res. 2008; 1(1): 9. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "53895", "date": "16 Oct 2019", "name": "Hafiz Farhan Maqbool", "expertise": [ "Reviewer Expertise Lower Limb Biomechanics", "Biomechatronics" ], "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\nMajor Comments:\nPage 5: Column 2: Instrumentation Section: It has been mentioned that the VICON system measures spatiotemporal parameters and hip, knee and ankle active range of motion during gait. The VICON system is normally used for capturing the data. For measuring and analyzing the kinematic parameters, there are other standard tools available such as Visual 3D. How the above-mentioned parameters are measured using VICON? Please justify it.\n\nHow can we impose a specific velocity such as 100 beats per minute? Can we impose this speed on any other population i.e. other than DMII? Perhaps, it is a big concern.\n\nThe study is evaluated while walking on an inclined surface only. What about the other activities such as stair ascending and descending and/or other complex activities? Can we impose the velocity of 100 bpm for other activities or even with different inclination angles?\n\nThe other concern is regarding the applicability of Mo-Cap data (through the VICON system or Kinect) on rehabilitation exercises. It can be argued that for rehab, one necessarily needs muscle-activity, and therefore, only movement data (i.e. without muscle activity) will be insufficient to conclusively comment on performance. Hence, try to incorporate muscle-activity e.g. using wearable surface EMG, if possible.\n\nThe results must also include a comparison with the existing literature. Perhaps, a comparison in a tabular form should be included.\nMinor Comments:\nPage 1: Abstract; Results: temporospatial and kinetic parameters…..In methods, temporospatial and kinematic parameters? Please clarify which statement is true. Also, generally the term spatiotemporal is used instead of temporospatial in the literature as you mentioned on page 5 instrumentation section.\n\nPage 1: Abstract: Conclusion is not clear.\n\nPage 3: Column 1: Introduction: Paragraph 1: BMI has not been defined.\n\nPage 5: Column 1: Line 1: AROM has not been defined.\n\nPage 5; Column 1: Line 2: …highly instrumented nature and the novelty of this study…..this sentence is not clear. Please rephrase it.\n\nPage 5: Column 2: Instrumentation section: …….recording at 120 Hz. The sentence is not clear. Also, it has been repeated twice within the same section.\n\nEnglish/Grammar of the manuscript needs to be improved.\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?\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? Partly", "responses": [] }, { "id": "66771", "date": "14 Jul 2020", "name": "Matthew K. Abramowitz", "expertise": [ "Reviewer Expertise Chronic kidney disease", "physical function", "gait abnormalities." ], "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 sample size of the study must be clarified. The number analyzed as indicated in Figure 1 and Table 1 is unclear. Figure 1 indicates 15 people in each of the DM and Control groups participated in the study and were analyzed, but also that 4 participants in the DM group and 5 in the Control group were excluded. Table 1 then presents data for 15 in each group.\n\nAlthough no statistically significant differences were noted, the knee and ankle kinematic data were numerically higher in the DM group, and the SDs indicate sizable variability in both groups. One wonders if these could be meaningful differences, and if they would be statistically significant if a larger sample size had been examined.\n\nThe authors note prior work that has demonstrated gait abnormalities in diabetic patients without peripheral neuropathy. The manuscript would be improved by greater discussion placing the current results in context and consideration of why the current results differ from prior work.\n\nThe authors note that even in the absence of neuropathy diabetes has been associated with reduced gait speed, partly as “an adaptive mechanism to feel safer.” The authors should comment on whether the lack of difference in spatiotemporal characteristics could be due to the imposed speed in their study design overcoming this adaptive mechanism.\n\nThe small sample size should be acknowledged as a limitation.\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": [] } ]	1	https://f1000research.com/articles/7-408

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